Antibodies for apoptosis EI24 protein and methods of use

ABSTRACT

Disclosed is the isolation and characterization of EI24, a novel gene whose 2.4 kb mRNA is induced following etoposide treatment. Induction of EI24 mRNA by etoposide required expression of wild-type p53. Overexpression of functional p53 was sufficient to induce expression of the EI24 mRNA. The EI24 mRNA was also induced in a p53-dependent manner by ionizing irradiation of primary murine thymocytes. The invention is thus directed to an isolated EI24 protein, nucleotide sequences coding for and regulating expression of the protein, antibodies directed against the protein, and recombinant vectors and host cells containing the genetic sequences coding for and regulating the expression of the protein sequence. The invention is also directed to genomic DNA, cDNA, and RNA encoding the EI24 protein sequence and to corresponding antisense RNA sequences. Antibodies can be used to detect EI24 in biological specimens, including, for example, human tissue samples. The present invention is further directed to methods of treating degenerative disorders characterized in inappropriate cell proliferation or inappropriate cell death. The present invention is further directed to methods for diagnosing degenerative disorders characterized in inappropriate cell proliferation or inappropriate cell death, as well as methods for monitoring the progress of such degenerative disorders.

FIELD OF THE INVENTION

[0001] The present invention relates generally to the field of cellphysiology, and more particularly, to apoptosis. Even more particularly,the present invention is related to a novel apoptosis associated proteinEI24 and its corresponding gene EI24; to nucleotide sequences encodingEI24; to products and processes involved in the cloning, preparation andexpression of genes and nucleotide sequences encoding EI24; toantibodies with specificity to EI24; and to diagnostic and therapeuticuses of the above.

BACKGROUND OF THE INVENTION

[0002] “Apoptosis” refers to cell suicide that proceeds by an active,physiological process (Kerr, J. F., et al., Br. J. Cancer 26:239-257(1972); Wyllie, A. H., Nature 284:555-556 (1980)). Apoptosis plays animportant role in developmental processes, including morphogenesis,maturation of the immune system, and tissue homeostasis whereby cellnumbers are limited in tissues that are continually renewed by celldivision (Ellis, R. E., et al., Annu. Rev. Cell. Biol. 7:663-698 (1991);Oppenheim, R. W., et al., Neurosci. 14:453-501 (1991); Cohen, J. J., etal., Annu. Rev. Immunol. 10:267-293 (1992); Raff., M. C., Nature356:397-400 (1992)).

[0003] In addition to its role in developmental processes, apoptosis isan important cellular safeguard against tumorigenesis (Williams, G. T.,Cell 65:1097-1098 (1991); Lane, D. P. Nature 362:786-787 (1993)).Defects in the apoptotic pathway may contribute to the onset orprogression of malignancies. Suppression of the apoptotic pathway(s), bya variety of genetic lesions, occurs frequently in a broad range ofhuman tumors. In particular, loss of the p53 tumor suppressor genefunction, either through deletion or mutation, occurs in more than 50%of human cancers. p53 gene function is also indicated in normal cellcycle events. Reviews of p53 function include Levine, A. J., et al.,Nature 351:453-456 (1991); Hollstein M., et al., Science 253:49-53(1991); Donehower, et al., Biochem. BioPhys. Acta 1155:181-205 (1993);Lane, D. P. Nature 362:786-787 (1993); Zambetti, et al., FASEB J.7:855-865 (1993); and Greenblatt M. S., et al., Cancer Res., 54:4855-4878 (1994).

[0004] p53 may exert its tumor suppressor function, at least in part, bydirecting cells that have sustained genomic damage to undergo apoptosis(Lowe S. W., Jacks T., Housman D. E. and Ruley H. E. (1994) Proc. Natl.Acad. Sci. USA, 91, 2026-2030). p53 is a sequence-specific DNA bindingprotein that functions both as a transcriptional activator and repressor(Donehower L. A. and Bradley A. (1993) Biochim. Biophys. Acta., 1155,181-205; Prives C. and Manfredi J. (1993) Genes Dev., 7, 529-534; FieldsS. and Jang S. K. (1990) Science, 249, 1046-1048; Raycroft L., Wu H. andLozano G. (1990) Science, 249, 1049-1051). Although there is someevidence that transcription may not be required in p53-mediatedapoptosis (Caelles C., Helmberg A. and Karin M. (1994) Nature, 370,220-223), several p53-regulated genes have been identified to date(Kastan M. B., Zhan Q., El-Deiry W. S., Carrier F., Jacks T., Walsh W.V., Plunkett B. S., Vogelstein B. and A. J. Fornace Jr. (1992) Cell, 71,587-597, 1992; El-Deiry W. S., Tokino T., Velculescu V. E., Levy D. B.,Parsons R., Trent J. M., Lin D., Mercer W. E., Kinzler K. W. andVogelstein B. (1993) Cell, 75, 817-825; Barak Y., Juven T., Haffner R.and Oren M. (1993) EMBO J., 12, 461-468; Wu X., Bayle J. H., Olson D.and Levine A. J. (1993) Genes & Dev., 7, 1126-1132; Zambetti G. P.,Bargonetti J., Walker K., Prives C. and Levine A. J. (1992) Genes &Dev., 6, 1143-1152; Okamoto K. and Beach D. (1994) EMBO J, 13,4816-4822; Buckbinder L., Talbott R., Seizinger B. R. and Kley N. (1994)Proc. Natl. Acad. Sci. USA, 91, 10640-10644, and two of these genes,bcl-2 and bax (Miyashita T. and Reed J. (1995) Cell, 80, 293-299;Miyashita T., Krajewski S., Krajewska M., Wang H., Lin H., Hoffman B.,Lieberman K. and Reed J. (1994) Oncogene, 9, 1799-1805; Zhan Q., Fan S.,Bae I., Guillouf C., Liebermann D. A., O'Connor P. M. and A. J. FornaceJr. (1994) Oncogene, 9, 3743-3751), have been clearly implicated inapoptosis (Oltvai Z. and Korsmeyer S. (1994) Cell, 79, 189-192).

[0005] In addition to cancer, deregulation of apoptosis may contributeto a number of other human diseases. A variety of degenerative disordersmay involve aberrant apoptosis, resulting in premature or inappropriatecell death (Barr, P. J., et al., Biotechnology 12:487-493 (1994))Productive infection by certain viruses may depend on suppression ofhost cell death by anti-apoptotic viral gene products (Rao, L., et al.,Proc. Natl. Acad. Sci. USA 89:7742-7746 (1992); Ray, C. A., et al., Cell69:597-604 (1992); White, E., et al., Mol. Cell. Biol. 12:2570-2580(1992); Vaux, D. L., et al., Cell 76:777-779 (1994), and inhibition ofapoptosis can alter the course (i.e. lytic vs. latent) of viralinfection; Levine, B., et al., Nature 361:739-742 (1993)). Widespreadapoptosis of T lymphocytes triggered by HIV infection may, at least inpart, be responsible for the immune system failure associated with AIDS(Gougeon M., et al., Science 260:1269-1270 (1993)).

[0006] The ability of p53 to suppress tumorigenesis appears linked toits activity as a transcriptional activator, since tumor-derived mutantp53 molecules almost invariably have lost transactivation potential(Kern, S. E., et al., Science 256:827-830 (1992)). Thus, the function ofthe p53 tumor suppressor appears to depend, at least in part, on theability to activate the expression of one or more target genes. Genesactivated by p53 may in turn mediate one or more aspects of p53's tumorsuppressor function, which including cell cycle arrest and apoptosis,depending on the cellular context. Consistent with the notion, certainp53-activated genes identified to date have been implicated in cellcycle control (gadd45, cyclin G, p21/WAF) and at least one p53-activatedgene (bax) is linked to the regulation of apoptosis.

[0007] Tumor cells frequently have lost wild-type p53 function.Consequently, activation of p53 target genes and associated tumorsuppressor functions, such as cell cycle arrest and apoptosis, isdefective in cancer cells. Therefore, from the perspective ofpharmaceutical development, identification of genes which are regulated(e.g, induced or repressed) by p53 may permit development of agents thatactivate, restore or suppress p53-dependent tumor suppression functionssuch as apoptosis or cell cycle regulation, depending on the clinicalsetting.

SUMMARY OF THE INVENTION

[0008] In a broad aspect, the present invention is directed to a gene,termed EI24, whose RNA was induced in NIH3T3 cells following treatmentwith etoposide. Experiments performed with cells derived fromp53-deficient mice demonstrate that induction of the EI24 mRNA isdependent on expression of functional p53 in vertebrate cells includingetoposide-treated fibroblasts, and gamma-irradiated thymocytes.Expression of a conditional p53-fusion protein demonstrates thatactivation of wild-type p53 function in E1A and T24H-ras-transformedp53-deficient fibroblasts is sufficient for induction of the EI24 mRNA.

[0009] The novel EI24 gene and the corresponding EI24 protein of mammalshave thus been isolated and characterized, and are described in a numberof embodiments herein. The present invention thus relates to anapoptosis associated protein EI24, products and processes involved inthe cloning, preparation and expression of genes for EI24; antibodieswith specificity to EI24; and nucleotide probes corresponding to theEI24 nucleotide sequence or portions thereof. The EI24 polypeptide isuseful for producing antibodies thereto. The antibodies and probes areuseful for detecting and isolating EI24 in biological specimensincluding for example, cells from all human tissues including hearttissue, lung tissue, tumor cells, brain tissue, placenta, liver,skeletal muscle, kidney, and pancreas.

[0010] The present invention further relates to species homologs andviral homologs of EI24.

[0011] In a particular embodiment, the human EI24 gene has been cloned.The present invention thus relates to the cloning, identification,characterization and sequencing of cDNAs and genomic fragments whichencode the EI24 that is present in human cells. The present inventionfurther relates to a method for isolating EI24 partial clones usingpolymerase chain reaction (PCR) cloning, from diverse human tumor celllines.

[0012] According to the present invention, there are provided geneticsequences encoding EI24. The instant invention also provides forexpression vectors containing such genetic sequences, hosts transformedwith such expression vectors, and methods for producing the geneticallyengineered or recombinant EI24.

[0013] The present invention also provides antibodies which specificallyrecognize EI24.

[0014] The EI24 cDNA and recombinant protein are useful for makingantibodies which specifically recognize EI24. Such antibodies are usefulfor detecting and isolating EI24 in a biological specimen. The EI24protein is also useful as a mediator of p53 function, particularly, p53tumor suppression function, including p53 mediated apoptotic and/or cellcycle control function.

[0015] The present invention is further directed to methods for inducingor suppressing p53 mediated functions including apoptosis in individualssuffering from degenerative disorders characterized by inappropriatecell proliferation or inappropriate cell death, respectively.Degenerative disorders characterized by inappropriate cell proliferationinclude, for example, inflammatory conditions, cancer, includinglymphomas, genotypic tumors, etc. Degenerative disorders characterizedby inappropriate cell death include, for example, autoimmune diseases,acquired immunodeficiency disease (AIDS), cell death due to radiationtherapy or chemotherapy, etc.

[0016] The present invention also relates to methods for detecting thepresence of EI24 protein, as well as methods directed to the diagnosisof degenerative disorders, which disorders are associated with anincreased or decreased level of expression of EI24, as compared to theexpected level of EI24 expression in the normal cell population.

[0017] The present invention is further directed to methods formonitoring the progress of degenerative disorders associated withincreased or decreased levels of expression of EI24, by monitoring EI24expression.

[0018] The present invention also relates to methods for determiningwhether a disease/degenerative disorder is linked to abnormal EI24expression, as well as methods for determining the effect of overexpression or loss of expression of EI24 in animal models such astransgenic mice and/or homozygous null mice. Methods for determiningwhether a disease/degenerative disorder is linked to abnormal EI24expression include analyzing EI24 expression in diseased tissue ascompared to normal tissue by for example, Northern and/or Western blots,as well as by other assay methods readily chosen and employed by thoseof ordinary skill in the art.

[0019] The present invention also relates to therapeutic methods andcompositions for modulating apoptotic effects by administering EI24protein, or a mutant or hybrid thereof, or by modulating expression ofthe EI24 gene, to an individual suffering from a degenerative disordercharacterized by inappropriate cell proliferation or inappropriate celldeath in order to stabilize inappropriate cell proliferation (i.e.,induce apoptosis) or stabilize inappropriate cell death (i.e., suppressapoptosis), respectively, and/or in either case to restore normal cellbehavior.

[0020] The present invention further relates to functional equivalentsincluding functional fragments of EI24 and/or EI24.

[0021] The present invention is also directed to nucleotide probes whichcan be used to determine the presence of EI24 as well as to identify andisolate homologs including species homologs and viral homologs.

DESCRIPTION OF THE FIGURES

[0022]FIG. 1.

[0023] Northern blot analysis of etoposide-treated murine 3T3 cellsusing the 11G10 and 38T19 probes. Cells were treated with 50 μMetoposide and poly-A⁺ RNA was harvested at the times indicated. Northernblots were probed sequentially with the 11G10 probe, the 38T19 probe anda tubulin probe.

[0024]FIG. 2.

[0025] Complete nucleotide sequence of EI24 cDNA and the predictedprotein sequence. The coding region is shown from positions 84 to 1034.The sequence of the 11G10 probe, identified by differential display, isunderlined. The accession number for the EI24 sequence submitted toGenBank is U41751.

[0026]FIG. 3.

[0027] Alignment of the EI24 predicted amino acid sequence and theCELF37C12.2 sequence from C. elegans.

[0028]FIG. 4.

[0029] Expression of the EI24 RNA in primary human tissues. A northernblot containing 2.0 μg of poly-A⁺ RNA from the indicated tissues wasprobed with the entire coding region from the murine EI24 cDNA. The blotwas stripped and rehybridized with a probe for b-actin as a loadingcontrol.

[0030]FIG. 5 Northern blot showing induction of EI24 RNA in irradiatedthymocytes. Thymocytes isolated from wild-type (p53) or p53-deficient(p53) mice were left untreated, or irradiated, and total RNA washarvested after the times indicated. 10 μg of total RNA was loaded perlane, and the gel was stained with ethidium bromide to visualize theribosomal RNA bands (18S rRNA shown). RNA was transferred tonitrocellulose, and the blot was hybridized with the EI24 cDNA. The blotwas stripped and rehybridized with a probe for glyceraldehyde phosphatedehydrogenase (GAPDH) as a loading control.

[0031]FIG. 6.

[0032] Northern blot showing induction of EI24 RNA by p53 in E1A/rastransformed murine embryonic fibroblasts (MEFs). 1.0 μg of poly-A⁺ RNAwas loaded per lane. p53^(+/+) and p53^(−/−) MEFs were left untreated,or treated with 50 μM etoposide for 6 hours, as indicated (lanes 1-4).Alternately, p53^(−/−) MEFs expressing either empty vector (pBabe Puro),or a tamoxifen-regulatable p53 (p53ER™) were left untreated, or treatedwith 3.3 μM tamoxifen (TMX) for 16 hours (lanes 5-8). The northern blotwas hybridized sequentially with probes for EI24, p21/WAF1 and tubulin.

[0033]FIG. 7.

[0034] Human Expressed Sequence Tag (EST) DNA sequences having greaterthan 90% homology with EI24. The descriptions of the EST clones andtheir GenBank EST database accession numbers are found in Table I.

DETAILED DESCRIPTION OF THE INVENTION

[0035] Genetic Engineering of EI24, Functional Equivalents Thereof andMutants Thereof

[0036] This invention comprises amino acid sequences of EI24 or EI24mutants, genetic sequences coding for such amino acid sequences,expression vehicles containing the genetic sequences, hosts transformedtherewith and recombinant EI24 and antisense RNA produced by suchtransformed host expression. The invention further comprises antibodiesdirected against EI24 and/or fragments thereof or against EI24 mutants.

[0037] The process for genetically engineering such protein sequences,according to the invention, is facilitated through the cloning ofgenetic sequences which are capable of encoding the peptide and throughthe expression of such genetic sequences. As used herein, the term“genetic sequences” is intended to refer to a nucleic acid molecule(preferably DNA). Genetic sequences which are capable of encoding theproteins are derived from a variety of sources. These sources includegenomic DNA, cDNA, synthetic DNA, and combinations thereof. Thepreferred source of the genomic DNA or mRNA is human tissue includingheart, lung, tumor cells, brain, placenta, liver, skeletal muscle,kidney and pancreas. The mRNA may then be used to obtain cDNA bytechniques known to those skilled in the art. Probes may be synthesizedbased on the nucleotide sequence of EI24 by methods known in the art.

[0038] The EI24 protein or fragment genomic DNA of the invention may ormay not include naturally occurring introns. Moreover, such genomic DNAmay be obtained in association with the 5′ promoter region of the EI24protein gene sequences and/or with the 3′ transcriptional terminationregion. Further, such genomic DNA may be obtained in association withthe genetic sequences which encode the 5′ non-translated region of theEI24 protein mRNA and/or with the genetic sequences which encode the 3′non-translated region. To the extent that a host cell can recognize thetranscriptional and/or translational regulatory signals associated withthe expression of the mRNA and protein, the 5′ and/or 3′ non-transcribedregions of the native gene, and/or the 5′ and/or 3′ non-translatedregions of the mRNA, may be retained and employed for transcriptionaland translational regulation. EI24 protein genomic DNA can be extractedand purified from human tissue by means well known in the art (forexample, see Guide to Molecular Cloning Techniques, S. L. Berger, etal., eds., Academic Press (1987)).

[0039] Alternatively, mRNA can be isolated from any cell which producesor expresses the protein, and used to produce cDNA by means well knownin the art (for example, see Guide to Molecular Cloning Techniques, S.L. Berger, et al., eds., supra. Preferably, the mRNA preparation usedwill be enriched in mRNA coding for such EI24 protein, either naturally,by isolation from cells which are producing large amounts of theprotein, or in vitro, by techniques commonly used to enrich mRNApreparations of specific sequences, including for example sucrosegradient centrifugation, or PCR. cDNA can then be prepared for example,by reverse transcription. The cDNA can then be amplified by PCR usingsuitable primers.

[0040] For cloning into a vector, such suitable DNA preparations (eitherhuman genomic DNA or cDNA) are randomly sheared or enzymaticallycleaved, respectively, and ligated into appropriate vectors to form arecombinant gene (either genomic or cDNA) library. A DNA sequenceencoding the EI24 protein or its functional equivalents may be insertedinto a DNA vector in accordance with conventional techniques, includingblunt-ending or staggered-ending termini for ligation, restrictionenzyme digestion to provide appropriate termini, filling in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and ligation with appropriate ligases. Techniques for suchmanipulations are disclosed, for example, by Sambrook J., Fritsch E. F.and Maniatis T. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, N.Y., 1989, (In: MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratories, ColdSpring Harbor, N.Y., 2d. ed. (1989)), and are well known in the art.

[0041] Libraries containing the EI24 protein clones may be screened anda EI24 clone identified by any means which specifically selects for EI24protein DNA such as, for example, (a) by hybridization with anappropriate nucleic acid probe(s) containing a sequence specific for theDNA of this protein, or (b) by hybridization-selected translationalanalysis in which native mRNA which hybridizes to the clone in questionis translated in vitro and the translation products are furthercharacterized, or, (c) if the cloned genetic sequences are themselvescapable of expressing mRNA, by immunoprecipitation of a translated EI24or fragment product produced by the host containing the clone.

[0042] Oligonucleotide probes specific for the protein which can be usedto identify clones to this protein can be designed from knowledge of theamino acid sequence of the EI24 protein. The sequence of amino acidresidues in a peptide is designated herein either through the use oftheir commonly employed three-letter designations or by theirsingle-letter designations. A listing of these three-letter andone-letter designations may be found in textbooks such as Biochemistry,2ed., Lehninger, A., Worth Publishers, New York, N.Y. (1975). When theamino acid sequence is listed horizontally, the amino terminus isintended to be on the left end whereas the carboxy terminus is intendedto be at the right end. The residues of amino acids in a peptide may beseparated by hyphens. Such hyphens are intended solely to facilitate thepresentation of a sequence.

[0043] Because the genetic code is degenerate, more than one codon maybe used to encode a particular amino acid (Watson, J. D., In: MolecularBiology of the Gene, 3rd Ed., W. A. Benjamin, Inc., Menlo Park, Calif.(1977), pp. 356-357). The peptide fragments are analyzed to identifysequences of amino acids which may be encoded by oligonucleotides havingthe lowest degree of degeneracy. This is preferably accomplished byidentifying sequences that contain amino acids which are encoded by onlya single codon.

[0044] Although occasionally an amino acid sequence may be encoded byonly a single oligonucleotide sequence, frequently the amino acidsequence may be encoded by any of a set of similar oligonucleotides.Importantly, whereas all of the members of this set containoligonucleotide sequences which are capable of encoding the same peptidefragment and, thus, potentially contain the same oligonucleotidesequence as the gene which encodes the peptide fragment, only one memberof the set contains the nucleotide sequence that is identical to theexon coding sequence of the gene. Because this member is present withinthe set, and is capable of hybridizing to DNA even in the presence ofthe other members of the set, it is possible to employ theunfractionated set of oligonucleotides in the same manner in which onewould employ a single oligonucleotide to clone the gene that encodes thepeptide.

[0045] Using the genetic code (Watson, J. D., In: Molecular Biology ofthe Gene, 3rd Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1977)), oneor more different oligonucleotides can be identified from the amino acidsequence, each of which would be capable of encoding EI24 or a fragmentprotein thereof. The probability that a particular oligonucleotide will,in fact, constitute the actual protein coding sequence can be estimatedby considering abnormal base pairing relationships and the frequencywith which a particular codon is actually used (to encode a particularamino acid) in eukaryotic cells. Such “codon usage rules” are disclosedby Lathe, et al., J. Molec. Biol. 183:1-12 (1985). Using the “codonusage rules” of Lathe, a single oligonucleotide sequence, or a set ofoligonucleotide sequences, that contains a theoretical “most probable”nucleotide sequence capable of encoding the EI24 protein sequences isidentified.

[0046] The suitable oligonucleotide, or set of oligonucleotides, whichis capable of encoding a fragment of the EI24 protein gene (or which iscomplementary to such an oligonucleotide, or set of oligonucleotides)may be synthesized by means well known in the art (see, for example,Synthesis and Application of DNA and RNA, S. A. Narang, ed., 1987,Academic Press, San Diego, Calif.) and employed as a probe to identifyand isolate the cloned EI24 protein gene by techniques known in the art.Techniques of nucleic acid hybridization and clone identification aredisclosed by Maniatis, et al. (In: Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.(1982)); Berger, et al., (In: Guide to Molecular Cloning Techniques,Academic Press (1988)); Sambrook J., Fritsch E. F. and Maniatis T.Molecular Cloning: A Laboratory Manual; Cold Spring Harbor LaboratoryPress: Cold Spring Harbor, N.Y., 1989, (In: Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,2d ed. (1989); and by Hames, et al. (In: Nucleic Acid Hybridization, APractical Approach, IRL Press, Washington, D.C. (1985)), whichreferences are herein incorporated by reference. Those members of theabove-described gene library which are found to be capable of suchhybridization are then analyzed to determine the extent and nature ofthe EI24 protein encoding sequences which they contain.

[0047] To facilitate the detection of the desired EI24 or fragmentprotein DNA encoding sequence, the above-described DNA probe is labeledwith a detectable group or label. Such detectable group or label can beany material having a detectable physical or chemical property. Suchmaterials have been well-developed in the field of nucleic acidhybridization and in general most any label useful in such methods canbe applied to the present invention. Particularly useful are radioactivelabels, such as ³²P, ³H, ¹⁴C, ³⁵S, ¹²⁵I, or the like. Any radioactivelabel may be employed which provides for an adequate signal and has asufficient half-life. The oligonucleotide may be radioactively labeled,for example, by “nick-translation” by well-known means, as described in,for example, Rigby, et al., J. Mol. Biol. 113:237 (1977) and by T4 DNApolymerase replacement synthesis as described in, for example, Deen, etal., Anal. Biochem. 135:456 (1983).

[0048] Alternatively, polynucleotides are also useful as nucleic acidhybridization probes when labeled with a non-radioactive marker such asbiotin, an enzyme or a fluorescent or chemiluminescent group. See, forexample, Leary, et al., Proc. Natl. Acad. Sci., USA 80:4045 (1983);Renz, et al., Nucl. Acids Res. 12:3435 (1984); and Renz, M., EMBO J.6:817 (1983).

[0049] Thus, the actual identification of the EI24 protein sequencespermits the identification of a theoretical “most probable” DNAsequence, or a set of such sequences, capable of encoding such apeptide. By constructing an oligonucleotide complementary to thistheoretical sequence (or by constructing a set of oligonucleotidescomplementary to the set of “most probable” oligonucleotides), oneobtains a DNA molecule (or set of DNA molecules), capable of functioningas a probe(s) for the identification and isolation of clones containingthe EI24 protein gene.

[0050] In an alternative way of cloning the EI24 protein gene, a libraryis prepared using an expression vector, by cloning DNA or, morepreferably, cDNA prepared from a cell capable of expressing the EI24protein, into an expression vector. The library is then screened formembers which express the EI24 protein, for example, by screening thelibrary with antibodies to the EI24 protein.

[0051] Preferred EI24 clones according to the invention include thehuman EI24 cDNA clone designated pKSEI24 1-2 and the composite murineEI24 cDNA clone designated pKSEI24 cl.11. Clones pKSEI24 1-2 and pKSEI24cl.11 have been deposited under the Budapest Treaty with the AmericanType Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md.20852, on or before the filing date of the present application, and havebeen assigned the ATCC Accession Numbers ______ and ______,respectively.

[0052] The above discussed methods are, therefore, capable ofidentifying genetic sequences which are capable of encoding EI24proteins or fragments thereof. In order to further characterize suchgenetic sequences, and, in order to produce the recombinant protein, itis desirable to express the proteins which these sequences encode. Suchexpression identifies those clones which express proteins possessingcharacteristics of the EI24 proteins. Such characteristics may includethe ability to specifically bind antibody to the EI24 protein and theability to elicit the production of an antibody or antibodies which arecapable of binding to the EI24 protein.

[0053] Expression of EI24 Protein, Fragments Thereof, FunctionalEquivalents Thereof, and Mutants Thereof

[0054] To express the EI24 protein or a functional equivalent, or mutantthereof, transcriptional and translational signals recognizable by anappropriate host are necessary. The cloned EI24 encoding sequences,obtained, for example, through the methods described above, andpreferably in a double-stranded form, may be operably linked tosequences controlling transcriptional expression in an expressionvector, and introduced into a host cell, either prokaryotic oreukaryotic, to produce recombinant EI24 protein or a functionalequivalent thereof. Depending upon which strand of the EI24 encodingsequence is operably linked to the sequences controlling transcriptionalexpression, it is also possible to express EI24 antisense RNA or afunctional equivalent thereof.

[0055] Expression of EI24 in different hosts may result in differentpost-translational modifications which may alter the properties of theEI24. The present invention encompasses the expression of the EI24protein, or functional equivalent thereof, or EI24 mutant, inprokaryotic or eukaryotic cells, and particularly, eukaryotic expressionis preferred.

[0056] Preferred prokaryotic hosts include bacteria such as E. coli,Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc. The mostpreferred prokaryotic host is E. coli. Other enterobacteria such asSalmonella typhimurium or Serratia marcescens, and various Pseudomonasspecies may also be utilized. Under such conditions, the protein may notbe glycosylated. The procaryotic host must be compatible with thereplicon and control sequences in the expression plasmid.

[0057] To express the EI24 protein (or a functional equivalent thereof)or EI24 mutant in a prokaryotic cell (such as, for example, E. coli, B.subtilis, Pseudomonas, Streptomyces, etc.), it is necessary to operablylink the EI24 encoding sequence to a functional prokaryotic promoter.Such promoters may be either constitutive or, more preferably,regulatable (i.e., inducible or derepressible). Examples of constitutivepromoters include the int promoter of bacteriophage lambda, the blapromoter of the Beta-lactamase gene of pBR322, and the CAT promoter ofthe chloramphenicol acetyl transferase gene of pBR325, etc. Examples ofinducible prokaryotic promoters include the major right and leftpromoters of bacteriophage lambda (P_(L) and P_(R)), the trp, recA,lacZ, lacI, and gal promoters of E. coli, the alpha-amylase (Ulmanen,I., et al., J. Bacteriol. 162:176-182 (1985)) and the sigma-28-specificpromoters of B. subtilis (Gilman, M. Z., et al., Gene 32:11-20 (1984)),the promoters of the bacteriophages of Bacillus (Gryczan, T. J., In: TheMolecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), andStreptomyces promoters (Ward, J. M., et al., Mol. Gen. Genet.203:468-478 (1986)). Prokaryotic promoters are reviewed by Glick, B. R.,(J. Ind. Microbiol. 1:277-282 (1987)); Cenatiempo, Y. (Biochimie68:505-516 (1986)); and Gottesman, S. (Ann. Rev. Genet. 18:415-442(1984)).

[0058] Proper expression in a prokaryotic cell also requires thepresence of a ribosome binding site upstream of the gene-encodingsequence. Such ribosome binding sites are disclosed, for example, byGold, L., et al. (Ann. Rev. Microbiol. 35:365-404 (1981)).

[0059] Especially preferred eukaryotic hosts include vertebrate andparticularly mammalian cells either in vivo, in animals or in tissueculture.

[0060] Expression of the EI24 in eukaryotic hosts requires the use ofregulatory regions functional in such hosts, and preferably eukaryoticregulatory systems. A wide variety of transcriptional and translationalregulatory sequences can be employed, depending upon the nature of theeukaryotic host. The transcriptional and translational regulatorysignals can also be derived from the genomic sequences of viruses whichinfect eukaryotic cells, such as adenovirus, bovine papilloma virus,Simian virus, herpes virus, or the like. Preferably, these regulatorysignals are associated with a particular gene which is capable of a highlevel of expression in the host cell.

[0061] In eukaryotes, where transcription is not linked to translation,such control regions may or may not provide an initiator methionine(AUG) codon, depending on whether the cloned sequence contains such amethionine. Such regions will, in general, include a promoter regionsufficient to direct the initiation of RNA synthesis in the host cell.Promoters from heterologous mammalian genes which encode mRNA productcapable of translation are preferred, and especially, strong promoterssuch as the promoter for actin, collagen, myosin, etc., can be employedprovided they also function as promoters in the host cell. Preferredeukaryotic promoters include the promoter of the mouse metallothionein Igene (Hamer, et al., J. Mol. Appl. Gen. 1:273-288 (1982)); the TKpromoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); theSV40 early promoter (Benoist, et al. Nature (London) 290:304-310(1981)); in yeast, the yeast gal4 gene promoter (Johnston, et al. Proc.Natl. Acad. Sci. USA 79:6971-6975 (1982); Silver, et al., Proc. Natl.Acad. Sci. USA 81:5951-5955 (1984)) or a glycolytic gene promoter may beused.

[0062] As is widely known, translation of eukaryotic mRNA is initiatedat the codon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes the EI24 protein, or a functionalequivalent thereof, does not contain any intervening codons which arecapable of encoding a methionine. The presence of such codons resultseither in the formation of a fusion protein (if the AUG codon is in thesame reading frame as the EI24 encoding DNA sequence) or a frame-shiftmutation (if the AUG codon is not in the same reading frame as the EI24encoding sequence).

[0063] If desired, a fusion product of the EI24 may be constructed. Forexample, the sequence coding for the EI24 or fragment thereof may beliked to a signal sequence which will allow secretion of the proteinfrom or the compartmentalization of the protein in, a particular host.Such signal sequences may be designed with or without specific proteasesites such that the signal peptide sequence is amenable to subsequentremoval.

[0064] Transcriptional initiation regulatory signals can be selectedwhich allow for repression or activation, so that expression of theoperably linked genes can be modulated. Of interest are regulatorysignals which are temperature-sensitive, such that by varying thetemperature, expression can be repressed or initiated, or which aresubject to chemical regulation, e.g., by a metabolite. Also of interestare constructs wherein the EI24 mRNA and antisense RNA are provided in atranscribable form, but with different promoters or othertranscriptional regulatory elements such that induction of EI24 mRNAexpression is accompanied by repression of antisense RNA expression,and/or repression of EI24 mRNA expression is accompanied by induction ofantisense RNA expression.

[0065] Translational signals are not necessary when it is desired toexpress EI24 antisense RNA sequences.

[0066] If desired, the non-transcribed and/or non-translated regions 3′to the sequence coding for the EI24 protein can be obtained by theabove-described cloning methods. The 3′-non-transcribed region may beretained for its transcriptional termination regulatory sequenceelements; the 3′-non-translated region may be retained for itstranslation termination regulatory sequence elements, or for thoseelements which direct polyadenylation in eukaryotic cells. Where thenative expression control sequence signals do not functionsatisfactorily in the host cell, then sequences functional in the hostcell may be substituted.

[0067] The vectors of the invention may further comprise other operablylinked regulatory elements such as enhancer sequences, or DNA elementswhich confer tissue or cell-type specific expression on an operablylinked gene.

[0068] To transform a mammalian cell with the DNA constructs of theinvention many vector systems are available, depending upon whether itis desired to insert the EI24 DNA construct into the host cellchromosomal DNA, or to allow it to exist in an extrachromosomal form.

[0069] If the EI24 DNA encoding sequence and an operably linked promoterare introduced into a recipient eukaryotic cell as a non-replicating DNA(or RNA) molecule, which may either be a linear molecule or a closedcovalent circular molecule which is incapable of autonomous replication,then the expression of the EI24 protein may occur through the transientexpression of the introduced sequence.

[0070] Genetically stable transformants may be constructed with vectorsystems, or transformation systems, whereby EI24 DNA is integrated intothe host chromosome. Such integration may occur de novo within the cellor, in a preferred embodiment, be assisted by transformation with avector which functionally inserts itself into the host chromosome, forexample, with retroviral vectors, transposons or other DNA elementswhich promote integration of DNA sequences into chromosomes. A vector isemployed which is capable of integrating the desired gene sequences intoa mammalian host cell chromosome.

[0071] Cells which have stably integrated the introduced DNA into theirchromosomes are selected by also introducing one or more markers whichallow for selection of host cells which contain the expression vector inthe chromosome, for example, the marker may provide biocide resistance,e.g., resistance to antibiotics, or heavy metals, such as copper, or thelike. The selectable marker gene can either be directly linked to theDNA gene sequences to be expressed, or introduced into the same cell byco-transfection.

[0072] In another embodiment, the introduced sequence is incorporatedinto a plasmid or viral vector capable of autonomous replication in therecipient host. Any of a wide variety of vectors may be employed forthis purpose, as outlined below.

[0073] Factors of importance in selecting a particular plasmid or viralvector include: the ease with which recipient cells that contain thevector may be recognized and selected from those recipient cells whichdo not contain the vector; the number of copies of the vector which aredesired in a particular host; and whether it is desirable to be able to“shuttle” the vector between host cells of different species.

[0074] Preferred eukaryotic plasmids include those derived from thebovine papilloma virus, vaccinia virus, SV40, and, in yeast, plasmidscontaining the 2-micron circle, etc., or their derivatives. Suchplasmids are well known in the art (Botstein, et al., Miami Wntr. Symp.19:265-274 (1982); Broach, J. R., In: The Molecular Biology of the YeastSaccharomyces: Life Cycle and Inheritance, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., pp. 445-470 (1981); Broach, J. R.,Cell 28:203-204 (1982); Bollon, et al., J. Clin, Hematol. Oncol.10:39-48 (1980); Maniatis, T., In: Cell Biology: A ComprehensiveTreatise, Vol. 3, “Gene Expression,” Academic Pres, NY, pp. 563-608(1980)), and are commercially available. For example, mammalianexpression vector systems which utilize the MSV-LTR promoter to driveexpression of the cloned gene, and in which it is possible tocontransfect with a helper virus to amplify plasmid copy number, andintegrate the plasmid into the chromosomes of host cells, have beendescribed (Perkins, et al., Mol. Cell Biol. 3:1123 (1983); Clontech,Palo Alto, Calif.).

[0075] Once the vector or DNA sequence containing the construct(s) isprepared for expression, the DNA construct(s) is introduced into anappropriate host cell by any of a variety of suitable means, includingtransfection. After the introduction of the vector, recipient cells aregrown in a selective medium, which selects for the growth ofvector-containing cells. Expression of the cloned gene sequence(s)results in the production of the EI24 protein, or in the production of afragment of this protein. This expression can take place in a continuousmanner in the transformed cells, or in a controlled manner, for example,expression which follows induction of differentiation of the transformedcells (for example, by administration of bromodeoxyuracil toneuroblastoma cells or the like).

[0076] The expressed protein is isolated and purified in accordance withconventional conditions, such as extraction, precipitation,chromatography, affinity chromatography, electrophoresis, or the like.Preferred proteins expressed according to the invention are the productsof the clone pKSEI24 cl.11 and particularly of the clone pKSEI24 1-2,described herein.

[0077] EI24 can be purified by growing the transformed host cells undersuitable conditions which are well known in the art, the cells can beharvested and disrupted to extract total cellular protein. The proteincan then, for example, be placed on a sizing column such as sepharose oragarose beads, and proteins of the correct molecular weight can becollected. The predicted molecular weight of EI24 is described herein.

[0078] Further purification can be effected by use of an anti-EI24antibody. Such an antibody can be used to immunoprecipitate EI24proteins from the set of cellular proteins of the correct approximatemolecular weight. Such antibodies can, for example, be raised againstpolypeptides synthesized according to the sequence or subsequences ofthe sequence shown herein. Alternatively, the antibodies can be raisedagainst fusion proteins, which contain EI24 sequences as well as thoseof other proteins. After immunoprecipitation, the EI24 proteins can bereleased from the antibodies to provide a substantially pure preparationof EI24 protein.

[0079] The EI24 DNA coding sequences, of the present invention may beused to obtain EI24 antisense RNA genetic sequences, inasmuch as theantisense RNA sequence will be that sequence found on the oppositestrand of the strand transcribing the peptide core's mRNA. The antisenseDNA strand may also be operably linked to a promoter in an expressionvector such that transformation with this vector results in a hostcapable of expression of a EI24 antisense RNA in the transformed cell.Antisense RNA and its expression may be used to interact with anendogenous EI24 DNA or RNA in a manner which inhibits or repressestranscription or translation of the-EI24 genes in a highly specificmanner. Use of antisense RNA probes to block gene expression isdescribed, for example, in Lichtenstein, C., Nature 333:801-802 (1988).

[0080] Construction and Identification of Antibodies Raised AgainstEI24, Functional Equivalents, Fragments, Hybrids, or Mutants Thereof

[0081] In the following description, reference will be made to variousmethodologies well-known to those skilled in the art of immunology.Standard reference works setting forth the general principles ofimmunology include the work of Catty, D., (Antibodies, A PracticalApproach, Vol. 1, IRL Press, Washington, D.C. (1988)); Klein, J.,(Immunology: The Science of Cell-Noncell Discrimination, John Wiley &Sons, New York (1982)); Kennett, et al., (Monoclonal Antibodies,Hybridoma: A New Dimension in Biological Analyses, Plenum Press, NewYork (1980)); Campbell, A. (“Monoclonal Antibody Technology,” In:Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13(Burdon, R., et al., eds.), Elsevier, Amsterdam (1984)); and Eisen, H.N. (In: Microbiology, 3rd ed. (Davis, B. D., et al., Harper & Row,Philadelphia (1980)).

[0082] An antibody is said to be “capable of binding” a molecule if itis capable of specifically reacting with the molecule to thereby bindthe molecule to the antibody. The term “epitope” is meant to refer tothat portion of a hapten which can be recognized and bound by anantibody. An antigen may have one, or more than one epitope. An“antigen” is capable of inducing an animal to produce antibody capableof binding to an epitope of that antigen. The specific reaction referredto above is meant to indicate that the antigen will react, in a highlyselective manner, with its corresponding antibody and not with themultitude of other antibodies which may be evoked by other antigens.

[0083] The term “antibody” (Ab) or “monoclonal antibody” (Mab) as usedherein is meant to include intact molecules as well as fragments thereof(such as, for example, Fab and F(ab)₂ fragments) which are capable ofbinding an antigen. Fab and F(ab)₂ fragments lack the Fc fragment ofintact antibody, clear more rapidly from the circulation, and may haveless non-specific tissue binding of an intact antibody (Wahl, et al., J.Nucl. Med. 24:16-325 (1983)).

[0084] The antibodies of the present invention have specificity to oneor more epitopes present on the EI24 peptide, or an idiotype thereon.The antibodies of the invention can be polyclonal or monoclonal,provided that they are made with the EI24 polypeptide or fragmentthereof as the immunogen. Both of these types of antibodies can beutilized in the applications described herein.

[0085] The present antibodies can be used to detect the presence of theEI24 protein in a human tissue sample. The EI24 protein can be detectedby contacting the sample with an imaging-effective amount of thedetectably labeled appropriate antibody and detecting the label, therebyestablishing the presence of the EI24 protein in the sample. Detectioncan be carried out by imaging in vivo. The EI24 protein can also bedetected by known immunoassay techniques, including, for example, RIA,ELISA, etc., using appropriate antibodies according to the invention.

[0086] The antibodies of the present invention are prepared by any of avariety of known methods. For example, cells expressing the EI24 proteincan be administered to an animal in order to induce the production ofserum containing polyclonal antibodies that are capable of binding theEI24 protein. For example, the EI24 protein or fragment thereof ischemically synthesized and purified by HPLC to render it substantiallyfree of contaminants. Such a preparation is then introduced into ananimal in order to produce polyclonal antisera of high specificactivity.

[0087] Polyclonal antibodies can be generated in any suitable animalincluding, for example, mice, rabbits or goats. The EI24 immunogenicpeptide or fragment thereof can be injected by itself or linked toappropriate immunoactivating carriers, such as Keyhole's limpethemocyanin (KLH). See Antibodies, A Practical Handbook, Vols. I and II,D. Catty, ed., IRL Press, Washington, D.C. (1988).

[0088] Monoclonal antibodies can be prepared in various ways usingtechniques well understood by those having ordinary skill in the art.For example, monoclonal antibodies can be prepared using hybridomatechnology (Kohler, et al., Nature 256:495 (1975); Kohler, et al., Eur.J. Immunol. 6:511 (1976); Kohler, et al., Eur. J. Immunol. 6:292 (1976);Hammerling, et al., In: Monoclonal Antibodies and T-Cell Hybridomas,Elsevier, N.Y., pp. 563-681 (1981)); Monoclonal Antibodies—Hybridomas: ANew Dimension in Biological Analysis, edited by Roger H. Kennett, etal., published by Plenum Press (1980). In general, such proceduresinvolve immunizing an animal with the EI24 protein, or a fragmentthereof. The splenocytes of such animals are extracted and fused with asuitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present invention. After fusion, theresulting hybridoma cells are selectively maintained in HAT medium, andthen cloned by limiting dilution as described by Wands, et al.,Gastroenterology 80:225-232 (1981). The hybridoma cells obtained throughsuch a selection are then assayed to identify clones which secreteantibodies capable of binding the EI24 protein.

[0089] Through application of the above-described methods, additionalcell lines capable of producing antibodies which recognize epitopes ofthe EI24 protein can be obtained.

[0090] For example, additional hybridomas which produce monoclonalantibodies which enable the detection of the EI24 protein can be easilyproduced and isolated with minimal screening. Hybridomas producingmonoclonal antibodies specific for epitopes which are found on the EI24protein are most effectively produced by first immunizing an animal fromwhich hybridomas can be produced such as, for example, a Balb/c mouse,with initial subcutaneous injections of Freund's adjuvant, followed bybooster injections within a few days. The fusion can be carried outusing any of the techniques commonly known to those of ordinary skill inthe art. The screening of the hybridomas to determine which ones areproducing monoclonal antibodies specific for a peptide isstraightforward and can be accomplished in a standard ELISA or RIAformat. For example, in an RIA screening format the culture supernatant,or ascites fluid from a hybridoma producing monoclonal antibody isreacted with ¹²⁵I-peptide. The isolation of other hybridomas secretingmAbs of the same specificity as those described herein can beaccomplished by the technique of anti-idiotypic screening. Potocmjak, etal., Science 215:1637 (1982). Briefly, an anti-idiotypic (anti-Id)antibody is an antibody which recognizes unique determinants generallyassociated with the antigen-binding site of an antibody. An Id antibodycan be prepared by immunizing an animal of the same species and genetictype (e.g., mouse strain) as the source of the mAb with the mAb raisedagainst the EI24 protein or fragment thereof to which an anti-Id isbeing prepared. The immunized animal will recognize and respond to theidiotypic determinants of the immunizing antibody by producing anantibody to these idiotypic determinants (the anti-Id antibody).

[0091] By using an anti-Id antibody which is specific for idiotypicdeterminants on a given mAb, it is then possible to identify other Bcell or hybridoma clones sharing that idiotype. Idiotypic identitybetween the antibody product of two clones makes it highly probable thatthe antibody products of the two clones recognize the same antigenicepitopes.

[0092] The anti-Id antibody may also be used as an “immunogen” to inducean immune response in yet another animal, producing a so-calledanti-anti-Id antibody. The anti-anti-Id may be epitopically identical tothe original mAb which induced the anti-Id.

[0093] Thus, by using antibodies to the idiotypic determinants of a mAb,it is possible to identify other clones expressing antibodies ofidentical specificity.

[0094] Accordingly, mAbs generated against the EI24 protein may be usedto induce anti-Id antibodies in suitable animals, such as BALB/c mice.Spleen cells from such immunized mice are used to produce anti-Idhybridomas secreting anti-Id mAbs. Further, the anti-Id mAbs can becoupled to a carrier such as keyhole limpet hemocyanin (KLH) and used toimmunize additional BALB/c mice. Sera from these mice will containanti-anti-Id antibodies that have the binding properties of the originalmAb specific for the antigen epitope. The anti-Id mAbs thus have theirown idiotypic epitopes, or “idiotopes” structurally similar to theepitope being evaluated.

[0095] For replication, the hybridoma cells of this invention may becultivated in vitro or in vivo. Production of high titers of mAbs invivo production makes this the presently preferred method of production.Briefly, cells from the individual hybridomas are injectedintraperitoneally into pristane-primed BALB/c mice to produce ascitesfluid containing high concentrations of the desired mAbs. MAbs ofisotype IgM or IgG may be purified from such ascites fluids, or fromculture supernatants, using column chromatography methods well known tothose of skill in the art.

[0096] Of special interest to the present invention are antibodies whichare produced in humans, or are “humanized” (i.e., non-immunogenic in ahuman) by recombinant or other technology such that they will not beantigenic in humans, or will be maintained in the circulating serum of arecipient for a longer period of time.

[0097] Humanized antibodies may be produced, for example by replacing animmunogenic portion of an antibody with a corresponding, butnon-immunogenic portion (i.e., chimeric antibodies) (Robinson, et al.,International Patent Publication PCT/US86/02269; Akira, et al., EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison, et al., European Patent Application 173,494;Neuberger, et al., PCT Application WO 86/01533, Cabilly, et al.,European Patent Application 125,023; Better, et al., Science240:1041-1043 (1988); Liu, et al., Proc. Natl. Acad. Sci. USA84:3439-3443 (1987); Liu, et al., J. Immunol. 139:3521-3526 (1987); Sun,et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Nishimura, et al.,Canc. Res. 47:999-1005 (1987); Wood, et al., Nature 314:446-449 (1985));Shaw, et al., J. Natl. Cancer Inst. 80:1553-1559 (1988). General reviewsof “humanized” chimeric antibodies are provided by Morrison, S. L.(Science, 229:1202-1207 (1985)) and by Oi, et al., BioTechniques 4:214(1986)).

[0098] Suitable “humanized” antibodies can be alternatively produced asdescribed by Jones, et al., Nature 321:552-525 (1986); Verhoeyan, etal., Science 234:1534 (1988), and Beidler, et al., J. Immunol.141:4053-4060 (1988).

[0099] The EI24 protein, fragments thereof, hybrids thereof, EI24mutants, or antibodies thereto can be utilized in immunoassays for thedetection of the EI24 protein in a human tissue sample. For example,antibodies against the EI24 protein can be used to detect the EI24protein in a human tissue sample. The immunoassays can be competitive orsandwich, as is otherwise well known and they all depend on theformation of antibody-antigen immune complex. These assays are wellknown to those of skill in the art.

[0100] For purposes of the assays, the antibody or antigen can beimmobilized or labeled. There are many carriers to which theantibody/antigen can be bound for immobilization and which can be usedin the present invention. Well-known carriers include but are notlimited to glass, polystyrene, polypropylene, polyethylene, dextran,nylon, amylases, natural and modified celluloses, polyacrylamides,agaroses, and magnetite. The nature of the carrier can be either solubleto some extent or insoluble for purposes of the invention. Those skilledin the art will know many other suitable carriers for binding theantibody or antigen, or will be able to ascertain such, using routineexperimentation.

[0101] Depending on the particular embodiment of the invention, one ormore of the antibodies or antigen(s) peptide(s) will be coupled with adetectable label such as an enzyme, radioactive isotope, fluorescentcompound, chemiluminescent compound, or bioluminescent compound.

[0102] Those of ordinary skill in the art will know of other suitablelabels for binding to the antibodies or antigen(s) peptide(s) or will beable to ascertain such using routine experimentation. Furthermore, thebinding of these labels to the antibodies or antigen(s) can be doneusing standard techniques commonly known to those of ordinary skill inthe art.

[0103] The antibodies or antigen peptide(s) can be bound to an enzyme.This enzyme, in turn, when later exposed to its substrate will reactwith the substrate in such a manner as to produce a chemical moietywhich can be detected, as, for example, by spectrophotometric orfluorometric means. Examples of enzymes that can be used to detectablylabel are amylate dehydrogenase, staphylococcal nuclease,delta-5-steroidisomerase, yeast alcoholdehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase, and acetylcholinesterase.

[0104] The presence of an antibody or antigen can also be detected bylabeling the antibody or antigen with a radioactive isotope. Thepresence of the radioactive isotope can be determined by such means asthe use of a gamma counter or a scintillation counter. Isotopes whichare particularly useful are ³H, ¹²⁵I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co,⁵⁹Fe, ⁷⁵Se, and ¹⁵²Eu.

[0105] It is possible to detect the presence of the antibody or antigenby labeling the antibody or antigen peptide with a fluorescent compound.When the fluorescently labeled antibody or antigen peptide is exposed tolight of the proper wavelength, its presence can then be detected due tofluorescence of the dye. Among the most common fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine.

[0106] Another way in which the antibody or antigen can be detectablylabeled is by coupling it to a chemiluminescent compound. The presenceof the chemiluminescent-tagged antibody or antigen peptide is thendetermined by detecting the presence of luminescence that arises duringthe course of a chemical reaction. Examples of particularly usefulchemiluminescent labeling compounds are luminol, isoluminol,aromatic-acridinium ester, imidaxole, acridinium salt, and oxalateester.

[0107] Likewise, a bioluminescent compound may also be used to label theantibody or antigen peptide. Bioluminescence is a special type ofchemiluminescence which is found in biological systems and in which acatalytic protein increases the efficiency of the chemiluminescentreaction. The presence of a bioluminescent binding partner would bedetermined by detecting the presence of luminescence. Importantbioluminescent compounds for purposes of labeling are luciferin,luciferase, and aequorin.

[0108] The antibodies or antigen peptide(s) for use in the assay of theinvention are ideally suited for the preparation of a kit. Such a kitmay comprise a carrier means being compartmentalized to receive in closeconfinement one or more container means such as vials, tubes, and thelike, each of said container means comprising one of the separateelements to be used in the method.

[0109] For example, one of the container means may comprise a firstantibody bound to an insoluble or partly soluble carrier. A secondcontainer may comprise soluble, detectably-labeled second antibody, inlyophilized form or in solution. The carrier means may also contain athird container means comprising a detectably labeled third antibody inlyophilized form or in solution. Such a kit can be used for sandwichassays.

[0110] In addition, the carrier means may also contain a plurality ofcontainers each of which comprises different, predetermined amounts ofthe EI24 peptide. These latter containers can then be used to prepare astandard curve into which can be used to interpolate the resultsobtained from the sample containing the unknown amount of the EI24protein.

[0111] Imaging can be carried out in vitro or in vivo. In vitro imagingcan be done with the labels mentioned previously. In vivo imaging isdone with diagnostically effective labeled antibodies. The term“diagnostically effective” means that the amount of detectably labeledantibody administered is sufficient to enable detection of the site ofEI24 protein presence when compared to a background signal.

[0112] Generally, the dosage of detectably-labeled antibody orantigen(s) for diagnosis will vary depending on considerations such asage, condition, sex, and extent of disease in the patient,counterindications, if any, and other variables, to be adjusted by theindividual physician. Dosage can very from 0.01 mg/kg to 2,000 mg/kg,preferably 0.1 mg/kg to 1,000 mg/kg.

[0113] The term “diagnostically labeled” means that the antibodyhas-attached to it a diagnostically detectable label.

[0114] There are many different imaging labels and methods of labelingknown to those of ordinary skill in the art. Examples of the types oflabels which can be used in the present invention include radioactiveisotopes and paramagnetic isotopes.

[0115] For diagnostic in vivo imaging, the type of detection instrumentavailable is a major factor in selecting a given radionuclide. Theradionucleotide chosen must have a type of decay which is detectable fora given type of instrument. In general, any conventional method forvisualizing diagnostic imaging can be utilized in accordance with thisinvention.

[0116] Another important factor in selecting a radionuclide for in vivodiagnosis is that the half-life of a radionucleotide be long enough sothat it is still detectable at the time of maximum uptake by the target,but short enough so that deleterious radiation upon the host isminimized. Ideally, a radionuclide used for in vivo imaging will lack aparticulate emission, but produce a large number of photons in a 140-200ke V range, which may be readily detected by conventional gamma cameras.

[0117] For in vivo diagnosis, radionucleotides may be bound to antibodyor antigen either directly or indirectly by using an intermediaryfunctional group. Intermediary functional groups which are often used tobind radioisotopes which exist as metallic ions to antibody or antigenare diethylenetriaminepentaacetic acid (DTPA) andethlenediaminetetracetic acid (EDTA). Typical examples of metallic ionswhich can be bound to immunoglobulins are ^(99m)Tc, ¹²³I, ¹¹¹In, ¹³¹I,⁹⁷Ru, ⁶⁷Cu, ⁷²Ga, ⁸⁹As, ⁸⁹Zr, and ²⁰¹T1.

[0118] The antibodies used in the method of the invention can also belabeled with paramagnetic isotopes for purposes of in vivo diagnosis.Elements which are particularly useful (as in magnetic resonance imaging(MRI) techniques) in this manner include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and⁵⁶Fe.

[0119] Preparations of the imaging antibodies for administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents are propyleneglycol,polyethyleneglycol, vegetable oil such as olive oil and injectableorganic esters such as ethyloleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media, parenteral vehicles including sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers, such as those based on Ringer'sdextrose, and the like. Preservatives and other additives may also bepresent, such as, for example, antimicrobials, anti-oxidants, chelatingagents, and inert gases and the like. See, generally, Remington'sPharmaceutical Science, 16th ed. Mac Eds. 1980.

[0120] Of course, the expressed EI24 protein is an intracellularprotein. Accordingly, those of skill will recognize that in vivodiagnostic and therapeutic methods employing the antibodies of theinvention may require some mechanism by which such antibodies can detectEI24 in the cell. One such method is to introduce the antibodies orfragments thereof into the cell itself across the cell membrane. Thismay be accomplished, for example, by attaching the antibody to a ligandfor which the target cell contains receptor sites. The antibody can thusbe transported into the cell membrane or across the cell membrane alongwith the ligand. Suitable ligands include growth factors and cytokinesthat are internalized upon receptor binding. Suitable growth factorsinclude epidermal growth factor (EGF), tumor growth factor alpha(TGF-α), fibroblast growth factor (FGF), insulin, and insulin-likegrowth factors 1 and 2 (TGF-1 and -2). Suitable cytokines include G-CSF,GM-CSF, erythropoietin, IL-1 and IL-2. It is noted that there are alsoreceptors that carry nutrients and vitamins into cells. These nutrientsare suitable for use as ligands in the present invention and includefoliate, dihdrofoliate, tetrahydrofoliate and vitamin B12.

[0121] The choice of a carrier ligand will depend on several factors, asthose of skill will appreciate. These include, for example, the kineticsof the ligand and its receptor, and of overall transport, which mayinclude passive or active, with actively transported ligands preferred.The means of attaching the antibody to the ligand also will vary withinlimits, and may be, for example, covalent or ionic, bearing in mind thatsuch attachment should not unacceptably alter ligand-receptor affinity.

[0122] Examples of receptors suitable for such applications include thereceptor for low density lipoprotein (LDL), which has been shown tocontain all the information necessary for receptor endocytosis, Davis etal., J. Cell Biol. 107(6/3): Abstr. No. 3112 (1988), as well as knownbrain-specific receptors such as those for dopamine. In this regard, itwill be appreciated that the ligand may itself be an antibody orfragment specific for the receptor, to which may be conjugated theantibody of the invention.

[0123] Moreover, those of skill may find it particularly desirable toemploy antibody fragments of the invention (such as, for example, Fab orF(ab′)₂ fragments), which are less likely to interfere with theligand-receptor interaction, and may be more easily transported acrossthe cell membrane. Single-chain antibodies may prove preferable forthese and other reasons, as will be appreciated by those of skill.

[0124] When an antibody is to be transported into the cell's membrane orinto the cell as described above, it will be preferred to diagnosticallyor therapeutically label the antibody in such a way that the label willbe relatively more effective when the antibody is bound to its antigenicsite on the EI24 protein. This may accomplished, for example, byemploying a label which becomes active or detectable as a result offormation of the antigen-antibody complex. Alternatively, the antibodyitself may be labeled in such a way that antigen-antibody complexformation induces a conformational change in the antibody to expose ormore fully expose the previously unexposed or less fully exposed label.All of the above criteria, and others, will be apparent to those ofskill in carrying out these aspects of the invention.

[0125] It is also possible to utilize liposomes having the antibodies ofthe present invention in their membranes to specifically deliver theantibodies to the target area. These liposomes can be produced so thatthey contain, in addition to the antibody, such therapeutic agents asdrugs, radioisotopes, lectins and toxins, which would act at the targetsite.

[0126] Pharmaceutical Compositions

[0127] Pharmaceutical compositions containing a therapeuticallyeffective amount of the EI24 protein, functional equivalents, fragmentsand/or hybrids and/or mutants thereof, as well as vectors containingcDNA encoding one or more of the foregoing, are useful for treatingpatients suffering from disorders in which p53 mediated functions suchas tumor suppression and cell cycle arrest are indicated, including butnot limited to degenerative disorders characterized by inappropriatecell death or inappropriate cell proliferation.

[0128] Hybrids of EI24 with one or more other proteins exhibit enhanced,decreased or intermediate effects on p53 mediated functions such asapoptosis induction or suppression activity as compared to the activityof EI24 alone. These hybrids can be readily selected, produced andemployed by one or-ordinary skill in the art. Pharmaceuticalcompositions according to the invention thus will contain atherapeutically effective amount of the EI24 protein, functionalequivalents, fragments and/or hybrids and/or mutants thereof, and mayoptionally contain one or more pharmaceutically acceptable carriersand/or excipients, known to those of ordinary skill in the art.Administration, dosage and frequency, and length of the course oftreatment can be readily optimized for a particular patient by one ofordinary skill in the art. For example, the present pharmaceuticalcomposition can be formulated as sterile aqueous or non-aqueoussuspensions or emulsions, as described above in Section IV, for examplefor solutions for intravenous administration.

[0129] Therapeutic Applications

[0130] The EI24 protein, functional equivalents, fragments and/orhybrids and/or mutants thereof as well as vectors containing cDNAencoding the foregoing are useful for treating cells, tissues or organsin which p53 mediated function is relevant to cell, tissue or organstate, including but not limited to states characterized byinappropriate cell death, inappropriate cell proliferation orinappropriate cell persistence. Particular disorders may involvedifferent cell types whereby it may be desirable to induce apoptosis inone cell type while suppressing apoptosis in the other. In addition,agents which modulate EI24 gene expression can be used therapeuticallyaccording to the present invention.

[0131] The therapeutic agents of the present invention can beadministered as discussed above with the requirement that the agent mustcross the cell membrane. The therapeutic agent can be administeredalone, in combination with or during the course of treatment with otheracceptable therapies known in the art for treating a particulardisorder. For example, the present therapeutic agents can beadministered to induce apoptosis in a cancer patient who is alsoundergoing classic cancer therapy including, for example, radiationtherapy, chemotherapy, and treatment with anti-cancer drugs including,for example, topoisomerase inhibitors, alkylating agents,antimetabolites, and hormone antagonists. Further, the presenttherapeutic agents can also be administered concurrently with genetherapy. For example, the present therapeutic agents can be administeredto a patient suffering from a degenerative disorder of the centralnervous system while the patient is concurrently undergoing gene therapyto replenish neutrophic hormones.

[0132] Premature widespread apoptosis (inappropriate cell death) causesmuch of the damage associated with degenerative disorders including, forexample, AIDs, chemotherapy and radiation, and tissue atrophy. In AIDspatients, lymphocytes are activated even in the asymptomatic phase ofthe HIV infection, and those cells die prematurely by apoptosis.

[0133] Those of skill will appreciate that administration of the variousproteins of the invention to particular target cells or tissues, asdescribed herein, is intended to comprehend the administration of theproteins themselves as well as the expression by the target cells ortissues of the nucleotide sequences encoding those proteins by variousknown means and in accordance with the teachings of the presentspecification. Methods for the in vivo treatment of vertebratesincluding humans at the gene level are known in the art and described,for example, in A. M. L. Lever and P. Goodfellow, Eds., “Gene Therapy,”British Medical Bulletin 51(1): 1-242, Churchill Livingstone, Pub.,Edinburgh (1995).

[0134] Degenerative disorders characterized in inappropriate cellproliferation include cancer, autoimmune disorders, tissue hypertrophy,and inflammatory disorders including inflammation arising from acutetissue injury including, for example, acute lung injury. Cancers arisewhen changes in DNA cause the anomalous accumulation of cells. Thecomparative rates of cell division and cell deaths determine how fast acancer grows. Some cancer cells divide more slowly than normal cells,but the cancer may still expand because of prolonged cell life span.Apoptosis is an efficient method for preventing malignant transformationbecause it removes cells with genetic lesions. Defective apoptosis canpromote cancer development, both by allowing accumulation of dividingcells and by obstructing removal of genetic variants with enhancedmalignant potential. The present therapeutic agents, as well as vectorscontaining cDNA encoding the one or more of the foregoing, can beadministered to cancer patients to induce apoptosis.

[0135] Many types of cancer can be treated by the administration of thepresent therapeutic agents, including for example, carcinomas, sarcomas,and leukemia/lymphomas, including for example, carcinomas such asadenocarcinomas, squamous carcinomas, carcinoma of the organs includingbreast, colon, head, neck, etc.; sarcomas including chondrosarcoma,melanosarcoma, etc.; and leukemia and lymphomas including acutelymphomatic leukemia, acute myelogenous leukemia, non-Hodgkin'slymphoma, Burkitt's lymphoma, B-cell lymphomas, T-cell lymphomas, etc.Other conditions amenable to treatment using the present therapeuticagent include fungal infections. Preferred according to the inventionare cancers in which p53 function is altered.

[0136] The present therapeutic agents can be used to treat autoimmunediseases. Random gene recombination and somatic hypermutation canpotentially generate autoreactive T and B lymphocytes throughout life.Under normal conditions immature lymphocytes that bind autoantigens dieby apoptosis. However, a defect in the deletion of these lymphocytespredisposes one to autoimmunity.

[0137] The present therapeutic agents can be administered to patientssuffering from autoimmune disorders to induce apoptosis in autoreactiveT lymphocytes, for example, in patients suffering systemic lupuserythematosus. Other autoimmune diseases amenable to treatment bysuppressing or inducing apoptosis through the administration of thepresent therapeutic agents include, for example, rheumatoid arthritis,myasthenia gravis, Grave's disease, Hashimoto's thyroiditis,insulin-resistent diabetes, allergic rhinitis, asthma, functionalautonomic abnormalities, juvenile insulin-dependent diabetes, Addison'sdisease, idiopathic hypoparathyroidism, spontaneous infertility,premature ovarian failure, pemphigus, Bullous pemphigoid, primarybiliary cirrhosis, autoimmune hemolytic anemia, idiopathicthrombocytopenic purpura, idiopathic neutropenia, Goodpasture'ssyndrome, rheumatoid arthritis and Sjogren's syndrome.

[0138] The present therapeutic agents can be used to treat inflammationresulting from acute lung injury, by inducing apoptosis. The diseaseprocess begins with an explosive inflammatory response in the alveolarwall. In the aftermath of the resulting tissue destruction, extensivefibroproliferation of the alveolar air space ensues, consisting offibroblasts, capillaries and their connective tissue products. Fukuda,Y., et al., Am. J. Pathol. 126:171-182 (1987). The present therapeuticagents can also be used to treat degenerative disorders due to prematureor excessive cell loss during aging which can lead to organ disfunctionand disease. Such degenerative disorders include degenerative diseasesof the central nervous system due to aging or other factors which resultin the death of neurons. The present therapeutic agents containing EI24mutant protein or hybrids thereof can be administered to a patientsuffering from such a degenerative disorder to suppress apoptosis.Further, the present therapeutic agents can be administered concurrentlywith gene therapy to provide genes encoding neutrophic hormonesincluding, for example, nerve growth factor. Other conditions amenableto treatment utilizing the present therapeutic agents include, forexample, Alzheimer's disease.

[0139] The present therapeutic agents can be administered to the desiredtarget cell as discussed below, for example, by choosing a receptor onthe target cell surface which is specific for that cell type. Thepresent therapeutic agents can be administered alone or in combinationwith other acceptable drug therapies. Further, the present therapeuticagents can be administered concurrently with other acceptable therapiesspecific for the particular degenerative disorder being treated. Forexample, the present therapeutic agents can be administered concurrentlywith chemotherapeutic agents, gene therapy, or the like. Therapeuticagents according to the invention must cross the cell membrane.

[0140] One method for introducing the therapeutic agents of theinvention into the cell's membrane or into the cell itself is byattaching the agent to a ligand for which the target cell containsreceptor sites. The agent can thus be transported into the cell membraneor across the cell membrane along with the ligand.

[0141] The choice of a carrier ligand will depend on several factors, asdiscussed herein and known to those of skill. Suitable tissue-specificreceptors include: Brain: nerve growth factor receptor (NGF-R); breast:prolactin receptor; stomach: gastrin receptor; skin: melanocytestimulating hormone receptor (MSH-R), liver: asialoglycoproteinreceptor; thyroid: thyroid stimulating hormone receptor (TSH-R);ovaries: luteinizing hormone receptor (LH-R), testis: human chorionicgonadotrophin receptor (hCG-R), T-cells: T-cell receptors; B cells:CD19; lung hyaluronate receptor CD44 isoform 4V (J. Cell. Biol. 124,7182, 1994). In this regard, it will be appreciated that the ligand maybe an antibody or fragment specific for the receptor, to which may beconjugated the EI24 protein of the invention.

[0142] It may be desirable to employ active EI24 fragments according tothe invention which are less likely to interfere with theligand-receptor interaction, and which may be more easily transportedacross the cell membrane.

[0143] When a protein is to be transported across the cell's membrane orinto the cell as described above and the ligand is an antibody, it willbe preferred to diagnostically or therapeutically label the protein insuch a way that the label will be relatively more effective when theprotein is bound, such as, for example, by means analogous to thosedescribed herein in the context of antibody transport.

[0144] It is also possible to utilize liposomes having the proteins ofthe present invention in their membrane to specifically deliver the EI24proteins to the target area. These liposomes can be produced so thatthey contain, in addition to the EI24 protein, such other therapeuticagents including drugs, radioisotopes, lectins and toxins, which wouldbe released at the target site.

[0145] A preferred manner for administering the EI24 encoding nucleotidesequences (and their functional equivalents and/or hybrids and/ormutants) for diagnostic or therapeutic purposes is by the use of viralvectors. Suitable viral vectors for gene transfer include retroviruses(reviewed in Miller, et al., Methods Enzymol. vol. 217, p.581-599(1993)) including human immunodeficiency virus (HIV), adenovirusderivatives (for examples see Erzurum, et al. Nucleic Acids Res. Vol.21, p.1607-12 (1993); Zabner, et al., Nat. Genet. Vol. 6, p.75-83(1994); Davidson, et al., Nat. Genet. vol. 3, p.219-223 (1993))adeno-associated virus (AAV), (i.e. see Flotte, et al., Proc. Natl.Acad. Sci. vol. 90, p.10613-7 (1993)) and Herpes virus vectors (i.e. seeAnderson, et al., Cell Mol. Neurobiol. vol. 13, p.503-15 (1993)). Othersuitable viruses can be readily selected and employed by those ofordinary skill in the art. Other methods for DNA delivery includeliposome mediated gene transfer (Alton, et al., Nat. Genet. vol. 5, p.135-42 (1993); Nabel, et al., Proc. Natl. Acad. Sci USA vol. 90, p.11307-11 (1993)).

[0146] The use of viral vectors for introduction of genes into mammaliancells is also reviewed, for example, in Varmus, Science 240(4858):1427(1988); Eglitis et al., BioTechniques 6, 7:608 (1988); Jaenisch, Science240(4858):1468 (1988); and Bernstein et al., Genet. Eng. (N.Y.) 7:235(1985).

[0147] For the purposes of the present invention, it may be preferred toemploy an attenuated viral or retroviral strain. Thus, for example, itis possible to use as vectors for the DNA sequences of the inventionretroviruses having attenuated cytopathicity, such as HIV-2_(ST) (Konget al., Science 240(4858):1525 (1988)) or HIV-2_(UC1) (Evans et al.,Science 240(4858):1523 (1988)), which enter neural cells by aCD4-dependent mechanism (Funke et al., J. Exp. Med. 165:1230 (1987)).The neurobiology of HIV infections is described, for example, in Johnsonet al., FASEB J. 2(14):2970 (1988). Those of skill will be able totarget different cell populations having known susceptibilities toviruses by the exercise of routine skill. For example, CD4 is known tohave a variant transcript in the human brain, with its highest contentin forebrain (Maddon et al., Cell 47:333 (1986). Possible methods totarget retroviral gene expression to specific cell types are reviewed byBoris-Lawrie and H. Temin Curr. Opin. Genet. Dev. vol. 3, p.102-9(1993).

[0148] Ideally, then, the choice of a gene delivery system will be madeby those of skill, keeping in mind the objectives of efficient andstable gene transfer, with an appropriate level of gene expression, in atissue-appropriate manner, and without any adverse effects. See, forexample, Wolff et al., Rheum. Dis. Clin. North Am. 14(2):459. (1988).With respect to delivery to a central nervous system target, many viralvectors, including HIV, offer the advantage of being able to cross theblood-brain barrier (Johnson et al., FASEB J. 2 (14):2970 (1988)).

[0149] Diagnostic Applications

[0150] Antibodies raised against the EI24 protein, fragments, functionalequivalents, or hybrids or mutants thereof can be used to detect theEI24 protein in a human tissue sample, as well as to diagnosedegenerative disorders associated with the expression of the EI24protein. Further, such antibodies can also be used to monitor theprogress of degenerative disorders associated with the expression of theEI24 protein.

[0151] Any source of human cells is suitable for use in the diagnostictesting in the present invention. The cells can be isolated from anyhuman tissue including for example, heart, lung, tumor cells, brain,placenta, liver, skeletal muscle, kidney and pancreas. Extraction ofproteins from the cell sample may be performed by any of the many meansknown in the art. For example, cells may be lysed by a detergent bymechanical means. If desired, nucleic acids can be removed from the cellpreparation by enzymatic digestion or by precipitation with agents suchas streptomycin. Such means are well known in the art.

[0152] Antibodies can be generated which are immunoreactive with theEI24 proteins by the methods set forth herein. Appropriate antibodiescan then be screened using the natural gene products of EI24.

[0153] The extracted proteins from the cell sample may be contacted withthe antibody under suitable conditions for antibody-antigen complexformation. Generally, such conditions are physiological conditions. Theprotein extract may be bound to a solid support such a nitrocellularfilter or a microtiter plate.

[0154] The antibody will generally bear a label which is a radio label,a florescent label, or an enzyme conjugate which under appropriateconditions produces, for example, a colored reaction product. Antibodiesand antibody labeling are described herein and known to those of skill.Alternatively, if the antibody is not labeled, it can be detected bymeans of a second antibody from another species which is reacted withthe first antibody. Suitable assay techniques, labels and means ofdetection are discussed herein.

[0155] A parallel sample to the test sample is employed to provide thecontrol. The control sample consists of an equivalent amount of proteinsextracted from cells, preferably in the same manner as those of the testsample. The amount of protein can readily be determined by employingtechniques well known in the art, including, for example, the Lowry orBradford techniques. The cells used for preparing the control sample maybe selected from cells of the same cell type as the test cells, isolatedfrom a normal human not suffering from the degenerative disorder fromwhich the human from which the test sample was taken suffers, cells ofthe same cell type as the test sample isolated from an establishednormal cell line, and cells from the human who is being tested, whichcell type is different from the cell type of the test cells.

[0156] Test samples can also be screened for elevated levels of mRNAtranscribed from the EI24 gene, according to methods well known in theart. For example, RNA extracted from B-cells may be used, oralternatively mRNA may be isolated from total cellular RNA The mRNA maybe purified, for example, by affinity chromatography on oligo (dTcellulose) which binds to the poly (A) tract at the 3′ end of most mRNA.As is well known to those skilled in the art, it is essential thatribonuclease activity be minimized during preparation and assaying.

[0157] A DNA probe may be selected from any of the protein codingsequences of the EI24 gene. Preferably, the probe will be selected fromsequences of the 5′ or 1st exon of the gene, so that all three speciesof RNA can be detected. Preferably, the probe contains at least 15nucleotides of the EI24 sequence. In order to perform the hybridization,it is desirable that the probe be single stranded. Thus, if the probe isdouble stranded, it should be denatured to a single stranded form. Meansfor denaturing are well known in the art, including alkali or heattreatment. The probe can then be contacted with the RNA derived from thecell sample under conditions where homologous RNA-DNA hybrids form andare stable. Such conditions are well known in the art. Means fordetecting hybrids are many and well known, but often involve the use ofradiolabeled probes and nucleases which degrade single stranded DNA.Other methods known in the art may be used.

[0158] Control samples can be derived from any of these cell sourcesdescribed above for use in the antibody diagnostic tests. Samples andcontrols should preferably be prepared in parallel under similarconditions.

[0159] The diagnostic methods and compositions of the present inventionare useful for determining whether a disease/degenerative disorder islinked to abnormal EI24 expression, as well as for determining theeffect of over expression or loss of expression of EI24 in animal modelssuch as transgenic mice and/or homozygous null mice. Methods fordetermining whether a disease/degenerative disorder is linked toabnormal EI24 expression include analyzing EI24 expression in diseasedtissue as compared to normal tissue by for example, Northern and/orWestern blots, as well as by other assay methods readily chosen andemployed by those of ordinary skill in the art. Once it has beendetermined that a disease/degenerative disorder is linked to abnormalEI24 expression, the disease/disorder can be diagnosed in an individual.

[0160] As used herein, the term “host” is meant to include not onlyprokaryotes, but also eukaryotes such as yeast and filamentous fungi, aswell as plant and animal cells. The term includes an organism or cellthat is the recipient of a replicable expression vehicle.

[0161] The term “substantially homologous” as used herein refers to theability of a first DNA sequence encoding EI24 to hybridize to a secondDNA sequence encoding the foregoing, under stringent conditions, forexample, at about 0.1×sodium citrate sodium chloride buffer (SSC) at atemperature of about 65° C.

[0162] The term “substantially pure” means that the protein or moleculeof interest is essentially free from any other detectable biologicalconstituents.

[0163] As used herein, a “functional equivalent” of the EI24 protein isa protein which possesses a biological activity or immunologicalcharacteristic substantially similar to a biological activity orimmunological characteristic of non-recombinant EI24. The term“functional equivalent is intended to include the “fragments,”“variants,” “analogues,” “homologues,” or “chemical derivatives” of amolecule which possess the biological activity of the EI24 protein ofthe invention.

[0164] A “fragment” of a molecule such as EI24 is meant to refer to anyvariant of the molecule which possess the biological activity of theEI24 protein. A “variant” of a molecule is meant to refer to a moleculesubstantially similar in structure and biological activity orimmunological characteristics to either the entire molecule, or to afragment thereof. Thus, provided that two molecules possess a similaractivity, they are considered variants as that term is used herein evenif the composition or secondary, tertiary, or quaternary structure ofone of the molecules is not identical to that found in the other, or ifthe sequence of amino acid residues is not identical. An “analog” of amolecule is meant to refer to a molecule substantially similar infunction to either the entire molecule or to a fragment thereof. As usedherein, a molecule is said to be a “chemical derivative” of anothermolecule when it contains additional chemical moieties not normally apart of the molecule. Such moieties may improve the molecule'ssolubility, absorption, biological half life, etc. The moieties mayalternatively decrease the toxicity of the molecule, eliminate orattenuate any undesirable side effect of the molecule, etc. Moietiescapable of mediating such effects are described, for example, inRemington's Pharmaceutical Sciences (1980). Procedures for coupling suchmoieties to a molecule are well known in the art.

[0165] By the term “anti-antibody” is intended an antibody directedagainst antigenic determinants on another antibody. By the term“anti-idiotypic antibody” is intended an antibody directed against anidiotypic determinant of another antibody. By the term “idiotope” isintended an idiotypic determinant, i.e., an antigenic determinant on avariable domain of an immunoglobulin molecule. By the term “idiotype” isintended a set of one or more idiotopes that distinguish a clone ofimmunoglobulin producing cells from other clones. Idiotypes occur in thevariable domains of immunoglobulin molecules and may be within, near to,or outside of the antigen binding site; antibodies to idiotypes locatedwithin or near the antigen biding site will prevent the immunoglobulinfrom combining with the antigen. By the term “idiotype-anti-idiotypenetwork” is intended a B-cell regulatory mechanism. Activation of a Bcell results in a clone of plasma cells producing immunoglobulin of asingle idiotype, which, because it was previously present in very smallquantities, can be recognized as “nonself” and results in the productionof anti-idiotypic antibodies directed against its idiotypicdeterminants. There can also be anti-anti-idiotypic antibodies directedagainst the second antibodies, antibodies directed against them, and soforth. These antibodies react with antigen receptors on B cells and Thelper and suppressor cells, as well as with circulating antibodies, toenhance or suppress production of the initial antibody by variousmechanisms.

[0166] By the term “administration” is intended any mode ofadministration which results in the delivery of the therapeutic agentacross the cell membrane and into the desired cell. The site ofadministration and cells will be selected by one of ordinary skill inthe art based upon an understanding of the particular degenerativedisorder being treated. In addition, the dosage, dosage frequency, andlength of course of treatment, can be determined and optimized by one ofordinary skill in the art depending upon the particular degenerativedisorder being treated. The particular mode of administration can alsobe readily selected by one of ordinary skill in the art and can include,for example, oral, intravenous, subcutaneous, intramuscular, etc., withthe requirement that the therapeutic agent cross the cell membrane. Thetherapeutic agent of the present invention can be the EI24 proteinand/or functional equivalents thereof and/or EI24 hybrids or EI24mutants and/or a vector containing cDNA encoding the foregoing.

[0167] By the term “therapeutic agent” is intended the EI24 protein,fragments, functional equivalents and/or hybrids or mutants thereof aswell as vectors containing cDNA encoding any of the foregoing. Thepresent therapeutic agent can be administered alone or in combinationwith and/or concurrently with other suitable drugs and/or courses oftherapy.

[0168] By the term “degenerative disorder” is intended for purposes ofthis invention, any disorder characterized by inappropriate cellproliferation, inappropriate cell death, inappropriate cell persistence,or in some cases, a compbinationof one or more of these. Preferredaccording to the invention are degenerative disorders in which p53function is altered. By the term “inappropriate cell proliferation” isintended a statistically significant increase in cell number as comparedto the proliferation of that particular cell type in the normalpopulation. Also included are disorders whereby a cell is present and/orpersists in an inappropriate location, e.g., the presence of fibroblastsin lung tissue after acute lung injury. For example, such cells includecancer cells which exhibit the properties of invasion and metastasis andare highly anaplastic. Such cells include but are not limited to, cancercells including, for example, tumor cells. By the term “inappropriatecell death” is intended a statistically significant decrease in cellnumber as compared to the presence of that particular cell type in thenormal population. Such under representation may be due to a particulardegenerative disorder, including, for example, AIDS (HIV), which resultsin the inappropriate death of T-cells, autoimmune diseases which arecharacterized by inappropriate cell death. By the term “inappropriatecell persistence” is intended a statistically significant persistence incell number as compared to the presence of that particular cell type inthe normal population. Such persistence may be due to p53 mediatedalterations in cell cycle arrest as compared to normal cells. By theterm “autoimmune disease” is intended a disorder caused by an immuneresponse directed against self antigens. Such diseases are characterizedby the presence of circulating autoantibodies or cell-mediated immunityagainst autoantigens in conjunctions with inflammatory lesions caused byimmunologically competent cells or immune complexes in tissuescontaining the autoantigens. Such diseases include systemic lupus,erythematosus (SLE), rheumatoid arthritis.

[0169] By the term “suppression” is intended for the purposes of thisinvention the result achieved by administering an amount of atherapeutic agent containing EI24 hybrids or EI24 mutants thereofeffective to suppress apoptosis in an individual suffering from adegenerative disorder characterized by inappropriate cell death.Suppression of apoptosis is achieved when the numbers of the particularaffected cell type remain stable or increase in number to a level withinthe range observed in the normal cell population. By the term “stable”is intended the state achieved when a statistically significant decreasein cell number is no longer observed in the individual being treated, ascompared to the cell number observed at the onset of the course oftreatment.

[0170] By the term “induction” is intended for the purposes of thisinvention the result achieved by the administration of an amount of atherapeutic agent containing the EI24 of the invention effective toinduce apoptosis in cells of an individual suffering from a degenerativedisorder characterized by inappropriate cell proliferation. Theinduction of apoptosis is achieved when cell numbers remain stable ordecrease to a level within the range observed in the normal cellpopulation. By the term “stable” is intended the state achieved duringthe course of treatment when a statistically significant increase incell proliferation is no longer observed as compared to the cell numberobserved at the onset of the course of treatment. One of ordinary skillin the art can readily determine whether the induction of apoptosis hasbeen achieved.

[0171] Particularly preferred uses of the compositions and methods ofthe present invention are those which involve p53 mediated function.Examples of such p53 mediated functions include but are not limited totumor suppression and cell cycle arrest, and are described, for examplein Levine, A. J., et al., Nature 351:453-456 (1991); Hollstein M., etal., Science 253:49-53 (1991); Donehower, et al., Biochem. BioPhys. Acta1155:181-205 (1993); Lane, D. P. Nature 362:786-787 (1993); Zambetti, etal., FASEB J. 7:855-865 (1993); and Greenblatt M. S., et al., CancerRes., 54: 4855-4878 (1994). EI24 expression appears to be affected byp53, and accordingly, preferred embodiments of the invention includethose in which this relationship is of diagnostic or therapeutic import.Particularly preferred according to the invention is the use of thecompositions of the invention in screening assays, as described herein,allowing the identification of agents which themselves are capable ofmediating p53 or EI24 function in cells which may be vertebrate cells,preferably mammalian cells, and more preferably, human cells.

[0172] By the term “normal cell behavior” is intended for the purposesof this invention, cells in which apoptosis proceeds normally. Normalcell behavior is observed in an organism which is able to removesenescent, damaged, or abnormal cells that could interfere with organfunction or develop into tumors. Apoptosis which proceeds normallyrepresents a coordinated cellular response to noxious stimuli that arenot immediately lethal.

[0173] By the term “patient” or “individual” is intended for thepurposes of the present invention, animals, including humans andmammals, who suffer from a degenerative disorder.

[0174] By the term “EI24 protein” is intended for the purposes of thepresent invention both the isolated naturally occurring and isolatedrecombinantly produced protein (i.e., synthetic EI24) which exhibits,inter alia, p53 induced expression in human tissue including, forexample, tumor cells and established human cell lines, and from tissuesof other invertebrate and vertebrate animals including mammals. Thisterm includes any analog, homolog, mutant or derivative of isolatednaturally occurring EI24 including fragments having less than thenaturally occurring number of amino acids, such as partial fragments ofnatural or synthetic EI24 which retain the biological or immunologicalcharacteristics of the polypeptide disclosed in this application. Thisterm also includes any peptide which contains the sequence of anisolated naturally occurring EI24 protein, or analog or homolog thereof,together with one or more flanking amino acids, which retains thebiological or immunological characteristics of the EI24 protein of theinvention.

[0175] The present invention pertains to both the expression offull-length EI24 and of functional derivatives of this protein,including allelic variants of EI24 and species or viral homologs ofEI24. Species homologs can be identified, isolated and recombinantlyproduced using the present nucleotide probes and procedures as describedherein, being methods well known in the art. Further, one of ordinaryskill in the art can readily determine whether a particular peptide is afunctional equivalent of EI24 using methods well known in the art.

[0176] More specifically, this term includes proteins encoded by thenucleotide sequence as shown in FIG. 2, and proteins having the aminoacid sequence as shown in FIG. 2, allelic variants, species homologs andviral homologs thereof, as well as functional derivatives thereofincluding fragments which retain the biological characteristics of EI24,and proteins that are substantially homologous thereto, which retain thebiological or immunological characteristics of the EI24 protein of theinvention.

[0177] The following Examples are offered by way of illustration, not byway of limitation.

EXAMPLES

[0178] Materials and Methods

[0179] Cell Culture. Murine NIH3T3 cells were cultured in DMEMsupplemented with 10% iron-fortified bovine calf serum, penicillin (5U/ml), streptomycin (50 μg/ml) and L-glutamine (4 mM). Etoposide (SigmaChemical Co., St. Louis, Mo.) was prepared as a 100 mM solution in DMSO,and used at a final concentration of 50 μM.

[0180] Differential display. Sub-confluent 3T3 cells were treated withetoposide (50 μM). Total cellular RNA was isolated by lysing cells withguanidinium thiocyanate, as described previously (Sambrook J., FritschE. F. and Maniatis T. Molecular Cloning: A Laboratory Manual; ColdSpring Harbor Laboratory Press: Cold Spring Harbor, N.Y., 1989). TotalRNA samples were treated with DNase I using the MessageClean kit(GenHunter, Brookline, Mass.) according to the manufacturer'sinstructions. Reverse transcription and PCR were performed as describedpreviously (Liang P. and Pardee A. B. (1992) Science, 257, 967-971),using the RNAmap kit (GenHunter). Four different 3′-primers (T₁₂NG,T₁₂NA, T₁₂NT, T₁₂NC) were used in combination with 20 different 10 bp5′-primers (AP1 to AP20) for PCR amplification, to generate a unique setof PCR products for each RNA population. PCR products were resolved ondenaturing polyacrylamide gels, and those that appeared to bedifferentially expressed were isolated from the gels, re-amplified, andlabeled with ³²P-dCTP for northern blot analysis. PCR products whichhybridized to differentially expressed RNA's, probe 11G10 (3′-primerT₁₂NG and 5′-primer AP10 or 5′-TAGCAAGTGC) and probe 38T19 (3′-primerT₁₂NT and 5′-primer AP19 or 5′-GGCTAATGCC), were cloned into pCR II,using the TA Cloning system (Invitrogen, San Diego, Calif.).

[0181] Northern blot analysis. Unless indicated otherwise, poly-A⁺ RNAwas extracted from cells using the Fast Track mRNA Isolation system(Invitrogen) according to the supplier's protocol. Northern blotanalysis was performed following standard methods (Sambrook J., FritschE. F. and Maniatis T. Molecular Cloning: A Laboratory Manual; ColdSpring Harbor Laboratory Press: Cold Spring Harbor, N.Y., 1989) using1.0 μg of poly-A⁺ RNA per lane. The northern blot containing poly-A⁺ RNA(2.0 μg) from normal human tissues was purchased from Clontech (PaloAlto, Calif.). Northern blots were probed with ³²P-labeled probes andexposed to X-ray film. Autoradiographs were scanned using an LKB 2202laser densitometer to obtain relative intensities of hybridizationsignal, The fold-increase or decrease in signal was normalized to thevalues obtained by hybridization to a control probe.

[0182] Isolation of a full length EI24 cDNA. A cDNA library wasgenerated in l-ZAP (Stratagene, La Jolla, Calif.) using poly-A⁺ RNAprepared from etoposide-treated 3T3 cells. Screening of this librarywith the probe generated by differential display, yielded one 850 bpclone, representing the 3′-end of the EI24 cDNA. The remaining 5′portion of the cDNA was isolated by screening a lgt11 3T3 librarypurchased from Clontech.

[0183] The extreme 5′ end of the EI24 cDNA was cloned using thePCR-based technique 5′-RACE, using the 5′-AmpliFINDER RACE kit(Clontech), according to the supplier's protocol. Poly-A⁺ RNA waspurified from etoposide-treated 3T3 cells. Two nested primers weredesigned; 11R8 (5′-GACTCACAAACATCCCCTGAATAAGG) was used for cDNAsynthesis, and 11R9 (5′-CTCCCTGATACTTCAAATGCCAAGTC) was used with theAnchor primer for PCR amplification. PCR amplification was carried outfor 35 cycles. The resulting PCR products were purified, and cloned intopCR II (above) for DNA sequence analysis. One 500 bp clone obtainedextended the largest 1gt11 clone by only 19 bp. The composite fulllength cDNA was assembled from the three separate clones intopBluescript KS II⁺ (Stratagene) and sequenced. The extreme 5′-end wasprovided by a 271 bp EcoRI-BamHI fragment isolated from the 5′-RACEclone. This was joined to a 1177 bp BamHI-ScaI fragment obtained fromthe largest 1gt11 clone Finally, the remaining 3′-untranslated regionwas cloned as a 709 bp ScaI-XhoI fragment from the single l-ZAP clone.The composite murine EI24 cDNA clone, designated pKSEI24 cl.11, has beendeposited with ATCC.

[0184] Isolation of a human EI24 cDNA. A cDNA encoding the human EI24homolog was cloned by PCR, relying on the high degree of homology tomurine EI24. Two oligonucleotide primers, 5′EI24M and 3′EI24, weresynthesized with the following DNA sequence: 5′EI24M:5′-CCCTCCATGATCAAAGCTTATGGTTTGGGGGCACTTCCCTCTAGCTGTATTTGATAGTCTGGGCAGTGGAGAGATG-3′3′EI24: 5′-TCTGTAAGCTTTGCTTTGCTTTAAAAAGACCACCAAGGAGAAGAGGCGC-3′

[0185] 5′EI24M corresponds to the first 19 amino acids encoded by themurine EI24 cDNA, which was determined to be sufficiently homologous tothe human cDNA to permit PCR amplification. 3′EI24 corresponds to the 3′end of the human EI24 open reading frame, based on partial EI24sequences deposited in the GenBank EST database.

[0186] A human EI24 cDNA clone was obtained by PCR amplication of cDNAprepared from Jurkat cells with the above primers. The PCR product wascloned into the HindIII site of pBluescript KS. This human EI@$ clone,designated pKSE124 1-2, has been deposited with ATCC.

[0187] Isolation of p53^(+/+) and p53^(−/−) thymocytes and Northern blotanalysis. Thymocytes were isolated from 4-week old wild-type andp53-null mice (C57BL/6×129/sv genetic background) (Jacks T., RemingtonL., Williams B. O., Schmitt E. M., Halachmi S., Bronson R. T. andWeinberg R. A. (1994) Current Biology, 4, 1-7) and placed in DMEsupplemented with 10% fetal bovine serum and 25 mM HEPES pH 7.2. Thecells were incubated at 37° C. following either no treatment or exposureto 600 cGy ionizing radiation. At the indicated times post treatment,total RNA was extracted from homogenized thymus tissue using the RNAzolB method according to manufacture's instructions (Biotecx Laboratories,Inc., Houston, Tex.). Approximately 10 mg of each RNA waselectrophoretically separated on a 1% agarose/1×MOPS/8.3% formaldehydegel. The RNAs were then transferred to nylon membranes in 20×SSC and UVcross linked to the membrane before prehybridizing, hybridizing andwashing as described previously (Shakleford G. and Varmus H. (1987)Cell, 50, 89-95). After hybridizing to a probe containing the entirecoding region of the EI24 cDNA, the blot was stripped and rehybridizedto a probe complementary to the glyceraldehyde phosphate dehydrogenase(GAPDH) mRNA, as a loading standard for each lane.

[0188] E1A and ras transformed mouse embryo fibroblasts and expressionof the p53-ER™ fusion protein. Stable cell lines of p53^(+/+) andp53^(−/−) MEFs expressing the oncogenes E1A and T24H-ras were generatedas described previously (Lowe S. W., Jacks T., Housman D. E. and RuleyH. E. (1994) Proc. Natl. Acad. Sci. USA, 91, 2026-2030). The plasmid forexpression of the p53ER™ fusion protein, pBabe Puro p53ER™ G525R, usingthe retroviral vector pBabe Puro (Morgenstern J. P. and Land H. (1990)Nucl. Acids Res., 18, 3587-3596), was received as a generous gift fromTrevor Littlewood (Imperial Cancer Research Fund, London). In brief,this plasmid contains the ˜1.3 kb BamHI fragment of human p53 frompSV53her (Roemer K. and Friedmann T. (1993) Proc. Natl. Acad. Sci. USA,90, 9252-9256) fused to the ligand-binding domain of the murine estrogenreceptor carrying a point mutation at amino acid 525 that renders thereceptor unable to bind estradiol but able to bind tamoxifen or4-hydroxy tamoxifen (Danielian P., White R., Hoare S., Fawell S. andParker M. (1993) Mol Endocrinol, 7, 232-240; Littlewood T. D., HancockD. C., Danielian P. S., Parker M. G. and Evan G. I. (1995) NAR, 23,1686-1690). For induction of wild-type p53 activity, p53ER™ transfectedp53^(−/−) cells were treated with 3.3 mM tamoxifen (Sigma).

[0189] Results

[0190] Identification of mRNAs for Etoposide-Responsive Genes.

[0191] Murine NIH3T3 fibroblasts were exposed to etoposide at aconcentration (50 μM) which was able to kill the majority ofasynchronously growing cells within 24 hours. Under these conditions,cells showed no obvious signs of apoptosis (by microscopic observation)for at least 6 hours, following exposure to etoposide. RNA was isolatedfrom 3T3 cells that had been treated with etoposide for 6 hours, andfrom non-treated cells. The two populations were compared by thedifferential display technique (Liang P. and Pardee A. B. (1992)Science, 257, 967-971), as described in the Materials and Methods. Atotal of 25 PCR products, which appeared to be differentially expressedin two independent experiments, were initially identified, and used asprobes in northern blots of mRNA from normal and etoposide-treated (6hours) 3T3 cells. By this analysis, only two of these probes proved tobe truly differentially expressed in the drug-treated versus untreatedcell populations (not shown). The PCR-derived probe designated 38T19identified a 5 kb mRNA whose expression was diminished inetoposide-treated cells; and probe 11G10 hybridized to a 2.4 kb mRNAthat increased in the etoposide-treated cells.

[0192] The differential expression of the mRNA species detected by thesetwo probes was examined more closely by northern blot analysis of 3T3cells exposed to etoposide for varying lengths of time (FIG. 1). The 2.4kb mRNA recognized by 11G10 was present at low levels in untreated cellsand was rapidly induced upon etoposide treatment, showing a 4-foldincrease by 3 hours post treatment. Induction of the 2.4 kb mRNAappeared to be maximal by 6 hours after etoposide exposure, representinga 7-fold increase. The same northern blot was stripped and re-hybridizedwith the 38T19 probe, which detected a 5 kb mRNA. The levels of thismRNA were highest in untreated cells, and decreased at 6 to 11 hoursafter etoposide treatment, corresponding to a 4-fold reduction.

[0193] The DNA sequence of probe 38T19, which detects the 5 kb mRNA, didnot significantly match any sequence in the nucleotide sequence database(not shown). Efforts to clone and characterize a full-length cDNAdetected by 38T19 are currently ongoing. We report here the cloning andcharacterization of the cDNA corresponding to the 2.4 kb mRNA recognizedby the 11G10 probe, named EI24, for Etoposide-Induced 2.4 kb mRNA.

[0194] Isolation of a Full-Length EI24 cDNA, and Analysis of itsSequence.

[0195] The 11G10 probe was used initially to isolate a partial (850 bp)EI24 cDNA clone from a cDNA library prepared from etoposide-treated 3T3cells. The remainder of the 2.4 Kb EI24 cDNA was isolated from a cDNAlibrary prepared from untreated 3T3 cells, with the exception of theextreme 5′-end of the cDNA (non-coding region, see below) which wasobtained by PCR methods. The DNA sequence of the composite 2118 bp EI24cDNA is shown in FIG. 2. An open reading frame extends from nucleotidepositions 84 to 1034, which codes for a 317 amino acid protein with apredicted molecular weight of 37 kD.

[0196] Comparison of the deduced sequence of the EI24 protein with theprotein sequence database revealed homology to the 316 amino acidsequence CELF37C12.2 (accession number U00033) from C. elegans. Asequence alignment of the two proteins is shown in FIG. 3. EI24 andCELF37 exhibit 25% homology overall, and 36% identity across the mostrelated region spanning amino acids 150 to 268. The CELF37 sequence wasdeduced by conceptual translation of C. elegans chromosome III DNAsequences (Wilson R., Ainscough R., Anderson K., Baynes C., Berks M.,Bonfield J., Burton J., Connell M., Copsey T., Cooper J., Coulson A.,Craxton M., Dear S., Du Z., Durbin R., Favello A., Fraser A., Fulton L.,Gardner A., Green P., Hawkins T., Hillier L., Jier M., Johnston L.,Jones M., Kershaw J., Kirsten J., Laisster N., Latreille P., LightningJ., Lloyd C., Mortimore B., O'Callaghan M., Parsons J., Percy C., RifkenL., Roopra A., Saunders D., Shownkeen R., Sims M., Smaldon N., Smith A.,Smith M., Sonnhammer E., Staden R., Sulston J., Thierry-Mieg J., ThomasK., Vaudin M., Vaughan K., Waterston R., Watson A., Weinstock L.,Wilkinson-Sproat J. and Wohldman P. (1994) Nature, 368, 32-38), and thefunction of this protein has not yet been determined. Neither EI24 norCELF37 showed significant homology to any other proteins in thedatabase.

[0197] Comparison of the DNA sequence of EI24 with the nucleotidesequence database identified several human cDNA sequences that exhibitedgreater than 90% homology to the EI24 cDNA (Table I). These sequences,which appear to represent portions of the human EI24 homologue, weresubmitted to the database as partial cDNA's (EST clones) isolated fromvarious human tissues. Neither the full cDNA sequence nor the functionof this human homologue has been reported. These data suggested to thepresent inventors that the EI24 mRNA may be widely expressed in normaltissues. To examine the expression pattern of the human EI24 homologue,the murine EI24 cDNA was used to probe a northern blot containing mRNAfrom a variety of primary human tissues. A 2.5 kb mRNA was detected inall tissues examined (FIG. 4), indicating that the human EI24 homologueis expressed in diverse tissues. TABLE I Human cDNA sequences havinggreater then 90% homology to the EI24 cDNA. Accession Number Descriptiongb/R17440 yg14b04.r1 Homo sapiens cDNA clone gb/T31497 EST33594 Homosapiens cDNA clone gb/H85229 yv85b08.r1 Homo sapiens cDNA clonegb/T99735 ye67e09.r1 Homo sapiens cDNA clone emb/Z25927 Homo sapienspartial cDNA clone gb/N36667 yx91d10.r1 Homo sapiens cDNA clonegb/N35767 yx81f05.r1 Homo sapiens cDNA clone

[0198] Induction of the EI24 RNA Requires p53.

[0199] To determine if the induction of EI24 is linked to a pathwayregulated by p53, we examined its expression in cells lacking p53function. p53 has been shown to play an essential role in thymocyteapoptosis in response to DNA damage (Clarke A. R., Purdie C. A.,Harrison D. J., Morris R. G., Bird C. C., Hooper M. L. and Wyllie A. H.(1993) Nature, 362, 849-852; Lowe S. W., Schmitt E. M., Smith S. W.,Osborne B. A. and Jacks T. (1993) Nature, 362, 847-849). We examinedwhether EI24 expression was induced in a p53-dependent manner in murinethymocytes exposed to ionizing radiation. Thymocytes derived from eitherwild-type or p53-deficient mice (see Materials and Methods) were treatedwith ionizing radiation and total RNA was isolated after 2 or 6 hours.The levels of EI24 expression were monitored by northern blot analysis(FIG. 5). While a low level of EI24 RNA was present in non-irradiatedthymocytes from both wild-type and p53-null mice, there was asubstantial induction of EI24 in wild-type, but not p53-deficient,tissue exposed to ionizing radiation. At 2 hours after treatment, therewas about a 2-fold increase in RNA levels, and at 6 hours aftertreatment the RNA was induced at least 7-fold (FIG. 5, lane 6). Theseresults are consistent with the pattern of EI24 expression inetoposide-treated 3T3 cells. It is noteworthy that when the northernblot was rehybridized with a GAPDH control probe, there wassignificantly lower signal in the wild-type 6-hours post irradiationsample; although the intensity of the 18S ribosomal RNA band in thissample was approximately the same as in the other lanes (FIG. 5, lane6). Given that a significant fraction of the cells were dead or dying atthis time point, we presume this reflects overall mRNA degradationduring apoptosis. Thus, the induction of the EI24 gene seen in thissample may be an under-estimate. These results demonstrate that ionizingradiation induces EI24 in thymocytes in a p53-dependent manner. p53 issimilarly required for etoposide induction of EI24 in transformedfibroblasts (see below).

[0200] EI24 mRNA is Induced by Over-Expression of p53 in TransformedFibroblasts.

[0201] Given that the EI24 mRNA was induced by two independentDNA-damaging agents thought to activate p53 (etoposide and ionizingradiation), we wondered whether elevation of p53 levels was sufficientto induce EI24 expression in p53-deficient cells. Murine embryonicfibroblasts (MEFs) obtained from a p53 “knockout” mouse (p53^(−/−)) orfrom a wild-type p53 mouse (p53^(+/+)), were transfected with adenovirusE1A and T24H-ras (Lowe S. W., Jacks T., Housman D. E. and Ruley H. E.(1994) Proc. Natl. Acad. Sci. USA, 91, 2026-2030). The resulting celllines were then treated with etoposide (50 μM) for 6 hours and northernblot analysis was performed as described above using poly-A⁺ RNA (FIG.6). No induction of EI24 was observed in etoposide-treatedE1A/ras-transformed p53^(−/−) cells, confirming that EI24 is induced byetoposide in a p53-dependent manner. The p53^(+/+) cells expressed a4-fold higher basal level of EI24 mRNA, even in the absence of etoposidetreatment, which was further induced in response to etoposide. The highbasal level of EI24 may reflect the action of wild-type p53 (see below)which is stabilized and present at higher levels in E1A/ras-transformedp53^(+/+) cells than in their untransformed counterparts (Lowe S. W. andRuley H. E. (1993) Genes & Dev., 7, 535-545).

[0202] The p53^(−/−) EIA/ras-transformed MEFs were then transfected witha p53-mutant estrogen receptor fusion protein (p53ER™). A similarp53-fusion, p53her, has been [Bdescribed previously and shown to bebiologically active (Roemer K. and Friedmann T. (1993) Proc. Natl. Acad.Sci. USA, 90, 9252-9256). In the case of p53ER™, the ligand bindingdomain of the murine estrogen receptor has been mutated to permitselective activation by the estrogen receptor antagonist tamoxifen(Littlewood T. D., Hancock D. C., Danielian P. S., Parker M. G. and EvanG. I. (1995) NAR, 23, 1686-1690; Danielian P., White R., Hoare S.,Fawell S. and Parker M. (1993) Mol Endocrinol, 7, 232-240). The p53ER™fusion is constitutively expressed as an inactive species, and onlyacquires wild-type p53 function in the presence of tamoxifen (C. A. V.et al. submitted for publication). Activation of p53 by the addition oftamoxifen for 16 hours induced expression of the 2.4 kb EI24 mRNA in thep53ER™-transfected p53^(−/−) cells, as detected by northern blotanalysis (FIG. 6). No EI24 induction was observed upon addition oftamoxifen to a matched p53^(−/−) control cell line (pBabe Puro) carryingthe empty vector alone.

[0203] Activation of the p53ER™ fusion by tamoxifen was confirmed byre-hybridizing the northern blot with a probe for p21/WAF1, a gene knownto be specifically induced by p53 (El-Deiry W. S., Tokino T., VelculescuV. E., Levy D. B., Parsons R., Trent J. M., Lin D., Mercer W. E.,Kinzler K. W. and Vogelstein B. (1993) Cell, 75, 817-825). Like EI24,the WAF1 mRNA was strongly induced upon tamoxifen treatment in thep53ER™ p53^(−/−) cells (FIG. 6, lanes 7 and 8), but not in the cellstransfected with the empty vector pBabe Puro (FIG. 6, lanes 5 and 6).Additionally, the basal (no etoposide) levels of WAF1 mRNA, like that ofEI24, were significantly elevated in E1A/ras transformed p53^(+/+) cells(FIG. 6, lane 1), reflecting the high levels of endogenous p53 expressedin these cells. In this example, activation of p53, either by tamoxifenin the p53ER™-transfected cells, or by its stabilization in the EIA andras-transformed p53^(+/+) cells, resulted in induction of both EI24 andWAF1 mRNAs. These results demonstrate that activation of p53 issufficient for induction of EI24, at least in the cell system describedhere.

[0204] Interestingly, the behavior of WAF1 RNA differed from that ofEI24 RNA in the p53^(−/−) MEFs which were treated with etoposide. WhileEI24 RNA was not induced by etoposide in these cells, WAF1 RNA wasinduced (see FIG. 6, lanes 3 and 4), although the maximal level ofinduction wa's lower than was seen in the p53^(+/+) cells. Induction ofWAF1 RNA through a p53-independent mechanism has been describedpreviously (Macleod K. F., Sherry N., Hannon G., Beach D., Tokino T.,Kinzler D., Vogelstein B. and Jacks T. (1995) Genes & Dev., 9, 935-944;Michieli P., Chedid M., Lin D., Pierce J., Mercer W. and Givol D. (1994)Cancer Res., 54, 3391-3395). The existence of a p53-independent pathwayfor apoptosis, triggered by etoposide (or irradiation), has also beendescribed (Strasser A., Harris A. W., Jacks T. and Cory S. (1994) Cell,79, 329-339) in p53^(−/−) lymphoma cells, and in proliferating T cellsderived from p53^(−/−) mice.

[0205] Discussion

[0206] The cytotoxicity of etoposide, as well as other chemotherapeuticdrugs, is dependent, at least in part, on its ability to triggerapoptosis through a p53-dependent pathway (Clarke A. R., Purdie C. A.,Harrison D. J., Morris R. G., Bird C. C., Hooper M. L. and Wyllie A. H.(1993) Nature, 362, 849-852; Lowe S. W., Ruley H. E., Jacks T. andHousman D. E. (1993) Cell, 74, 957-967). Etoposide-induced apoptosisrequires RNA synthesis (Walker P., Smith C., Youdale T., Leblanc J.,Whitfield J. and Sikorska M. (1991) Cancer Res., 51, 1078-1085), raisingthe possibility that one or more genes transactivated by p53 in responseto etoposide is involved in implementing the cell death program. TheEI24 gene, isolated and characterized in this study, is a candidate fora gene in this class based upon several properties of its expression.First, the EI24 mRNA is induced rapidly in response to etoposideexposure or ionizing radiation, with kinetics that precede or parallelthe onset of apoptosis. Second, induction of EI24 mRNA in response toDNA damage was only observed in cells expressing functional p53.Finally, over-expression of p53, in the absence of etoposide or otherDNA-damaging agents, results in induction of the EI24 gene.

[0207] The properties of EI24 expression are consistent with itspotential role in p53-mediated apoptosis. The function of EI24 inapoptosis, may be restricted to a p53-dependent cell death pathway,since expression of EI24 mRNA was not altered following induction ofapoptosis by either Fas ligation, or cytokine withdrawal in IL-3dependent cells. EI24 is, therefore, not induced by apoptotic stimuliper se. EI24 may contribute to a different p53-dependent process, suchas growth arrest or DNA repair.

[0208] Basal levels of EI24 expression appear independent of p53, whileits induction in response to DNA damage is regulated by p53. p53 maytransactivate EI24 either directly, by binding to sites within the EI24promoter, or indirectly, by regulating the expression of a gene whoseproduct in turn governs EI24 transcription.

[0209] The homology of the sequence of the EI24 protein to the C.elegans protein CELF37C12.2, implies an evolutionarily conservedfunction for EI24. There is substantial precedence for the structuraland functional conservation in C. elegans and mammals of genes thatregulate apoptosis (Steller H. (1995) Science, 267, 1445-1449). Examplesinclude the C. elegans ced3 and ced9 cell death regulatory genes, whosemammalian counterparts, IL-1-β-converting enzyme (ICE) and bcl-2,respectively, modulate apoptosis in a similar fashion. The homology ofEI24 to CELF37C12.2 raises the possibility that the regulation of EI24will likewise be conserved. If so, the CELF37C12.2 gene may, by analogyto EI24, also be induced in C. elegans in response to DNA damage. Thiswould suggest the existence of a conserved p53-like regulatory functionin nematodes and provides a means to identify such genes.

[0210] Publications mentioned in this specification are indicative ofthe level of the skill of those skilled in the art to which thisinvention pertains. All publications are herein incorporated byreference, to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

[0211] While the invention has been described in connection with thespecific embodiments thereof, it will be understood that it is capableof further modifications and this application is intended to cover anyvariations, uses, or adoptions of the invention following, in general,principles of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

1 21 1 10 DNA Artificial Sequence primer sequence 1 tagcaagtgc 10 2 10DNA Artificial Sequence primer sequence 2 ggctaatgcc 10 3 26 DNAArtificial Sequence primer sequence 3 gactcacaaa catcccctga ataagg 26 426 DNA Artificial Sequence primer sequence 4 ctccctgata cttcaaatgccaagtc 26 5 76 DNA Homo sapiens 5 ccctccatga tcaaagctta tggtttgggggcacttccct ctagctgtat ttgatagtct 60 gggcagtgga gagatg 76 6 49 DNA Homosapiens 6 tctgtaagct ttgctttgct ttaaaaagac caccaaggag aagaggcgc 49 72119 DNA Murine 7 tgaacccggg acgcgaaagg cggcaggggc aggcctggcg gcggcgcgcgggactcaggc 60 tttccccagg ccctccatga tgaatggttt gggggcactt ccctctagctgtatttgata 120 gtctgggcag tggagagatg gctgacagtg tcaaaacctt tctgcaggaccttggcaggg 180 gaatcaaaga ctccatctgg ggcatctgta ccatctcaaa gctagatgctcggatccagc 240 agaagagaga ggaacagcgt cgaagaaggg caagtagcct cttggcccagaggagacccc 300 agagtgtaga gcggaagcaa gagagtgaac cacgtattgt tagtagaattttccagtgtt 360 gtgcttggaa tggtggagta ttctggttca gtctcctctt gttttatcgagtgtttattc 420 ctgtacttca gtcagtaaca gcccggatta ttggagatcc atcacttcatggagatgttt 480 ggtcatggct ggaattcttc ctcacatcaa ttttcagtgc tctttgggtgctccccctgt 540 ttgtgcttag caaagttgtg aatgccattt ggttccaaga tatagctgacttggcatttg 600 aagtatcagg gaggaaacct catccattcc ccagtgtcag caaaataattgctgacatgc 660 tcttcaacct tttgctacag gcacttttcc ttattcaggg gatgtttgtgagtctcttcc 720 ccatccatct tgtgggtcag ctggttagtc tgctgcatat gtctcttctctattcactgt 780 actgctttga gtaccgttgg ttcaacaaag gaattgaaat gcaccagcgattgtcgaaca 840 tagaaaggaa ttggccttac tactttgggt ttggcttgcc cttggctttcctcacagcaa 900 tgcaatcctc ctacattatc agtggctgcc tcttttctat cctgtttcctttattcatca 960 tcagcgccaa tgaagcaaag actcctggaa aagcatatct tttccagttgcgcctattct 1020 ccttggtggt ctttttaagc aacagacttt tccacaagac cgtctacctgcagtcagccc 1080 tgagcagctc gtcctctgca gagaaattcc cttcgccaca tccttctccggccaaactga 1140 aagctgctgc aggccattga gccctgctgt caaaggggtg ggtgggactgggtggaggat 1200 gtggcagctc ttttctctgt tttcctcccc ctgccgtgga aggcagaacccactgccaag 1260 ggccctctgc atagtccctt gtctttgaat tggaatcttc ctgactccagtatatggatt 1320 tttaccacca ccctaggtct gtaaggacca gttttccagc tgtttttttagcacttgcca 1380 gctcctgtgc ctggactgat tgatttgagt actttttttc ccctttccttgtgtcattta 1440 ccctcccact tcctcctgcc ttccagcacc cctggatgaa tgggctttgtaattttaact 1500 gttgtatttt gtgaatttgt tgttactgtt tttctgtgaa gcacatacatgtatgtggga 1560 ggtaaagggg cattccagtt gctccagtca ctccctctat agccatactgtcttgttttc 1620 tgtaactcag gttaggtttt ggtctctatt ctctgctgca gaaaaggaaagaaggagtgg 1680 gggaaatgga gcctgaagag ttggggcaga tagacctcag ccaaactggctgggttttga 1740 ggagtcatgt tctttcttcc cttgaagggg aaagagtttt ttccactggtccatttaaag 1800 tttcccagct atggggtggt accagttctg gacaagtgcc actgcatcatagtatgctcg 1860 gagaatctga accttactct gaagatgaaa tttactgttg ccactgccaggtcacactgg 1920 tgttttaagg aatactgggt gcttcatata ggaactgaag gggtaaacttactaaaccat 1980 tcaacctgtg attggtgatg ttttcctgtc attttaagag tcgacacatgggtggggggg 2040 cagatgtaaa aaaacttgta caattttaaa atatcacaat taaacgtgagctggtttccc 2100 aaaaaaaaaa aaaaaaaaa 2119 8 358 PRT Murine 8 Met Val TrpGly His Phe Pro Leu Ala Val Phe Asp Ser Leu Gly Ser 1 5 10 15 Gly GluMet Ala Asp Ser Val Lys Thr Phe Leu Gln Asp Leu Gly Arg 20 25 30 Gly IleLys Asp Ser Ile Trp Gly Ile Cys Thr Ile Ser Lys Leu Asp 35 40 45 Ala ArgIle Gln Gln Lys Arg Glu Glu Gln Arg Arg Arg Arg Ala Ser 50 55 60 Ser LeuLeu Ala Gln Arg Arg Pro Gln Ser Val Glu Arg Lys Gln Glu 65 70 75 80 SerGlu Pro Arg Ile Val Ser Arg Ile Phe Gln Cys Cys Ala Trp Asn 85 90 95 GlyGly Val Phe Trp Phe Ser Leu Leu Leu Phe Tyr Arg Val Phe Ile 100 105 110Pro Val Leu Gln Ser Val Thr Ala Arg Ile Ile Gly Asp Pro Ser Leu 115 120125 His Gly Asp Val Trp Ser Trp Leu Glu Phe Phe Leu Thr Ser Ile Phe 130135 140 Ser Ala Leu Trp Val Leu Pro Leu Phe Val Leu Ser Lys Val Val Asn145 150 155 160 Ala Ile Trp Phe Gln Asp Ile Ala Asp Leu Ala Phe Glu ValSer Gly 165 170 175 Arg Lys Pro His Pro Phe Pro Ser Val Ser Lys Ile IleAla Asp Met 180 185 190 Leu Phe Asn Leu Leu Leu Gln Ala Leu Phe Leu IleGln Gly Met Phe 195 200 205 Val Ser Leu Phe Pro Ile His Leu Val Gly GlnLeu Val Ser Leu Leu 210 215 220 His Met Ser Leu Leu Tyr Ser Leu Tyr CysPhe Glu Tyr Arg Trp Phe 225 230 235 240 Asn Lys Gly Ile Glu Met His GlnArg Leu Ser Asn Ile Glu Arg Asn 245 250 255 Trp Pro Tyr Tyr Phe Gly PheGly Leu Pro Leu Ala Phe Leu Thr Ala 260 265 270 Met Gln Ser Ser Tyr IleIle Ser Gly Cys Leu Phe Ser Ile Leu Phe 275 280 285 Pro Leu Phe Ile IleSer Ala Asn Glu Ala Lys Thr Pro Gly Lys Ala 290 295 300 Tyr Leu Phe GlnLeu Arg Leu Phe Ser Leu Val Val Phe Leu Ser Asn 305 310 315 320 Arg LeuPhe His Lys Thr Val Tyr Leu Gln Ser Ala Leu Ser Ser Ser 325 330 335 SerSer Ala Glu Lys Phe Pro Ser Pro His Pro Ser Pro Ala Lys Leu 340 345 350Lys Ala Ala Ala Gly His 355 9 358 PRT Murine 9 Met Val Trp Gly His PhePro Leu Ala Val Phe Asp Ser Leu Gly Ser 1 5 10 15 Gly Glu Met Ala AspSer Val Lys Thr Phe Leu Gln Asp Leu Gly Arg 20 25 30 Gly Ile Lys Asp SerIle Trp Gly Ile Cys Thr Ile Ser Lys Leu Asp 35 40 45 Ala Arg Ile Gln GlnLys Arg Glu Glu Gln Arg Arg Arg Arg Ala Ser 50 55 60 Ser Leu Leu Ala GlnArg Arg Pro Gln Ser Val Glu Arg Lys Gln Glu 65 70 75 80 Ser Glu Pro ArgIle Val Ser Arg Ile Phe Gln Cys Cys Ala Trp Asn 85 90 95 Gly Gly Val PheTrp Phe Ser Leu Leu Leu Phe Tyr Arg Val Phe Ile 100 105 110 Pro Val LeuGln Ser Val Thr Ala Arg Ile Ile Gly Asp Pro Ser Leu 115 120 125 His GlyAsp Val Trp Ser Trp Leu Glu Phe Phe Leu Thr Ser Ile Phe 130 135 140 SerAla Leu Trp Val Leu Pro Leu Phe Val Leu Ser Lys Val Val Asn 145 150 155160 Ala Ile Trp Phe Gln Asp Ile Ala Asp Leu Ala Phe Glu Val Ser Gly 165170 175 Arg Lys Pro His Pro Phe Pro Ser Val Ser Lys Ile Ile Ala Asp Met180 185 190 Leu Phe Asn Leu Leu Leu Gln Ala Leu Phe Leu Ile Gln Gly MetPhe 195 200 205 Val Ser Leu Phe Pro Ile His Leu Val Gly Gln Leu Val SerLeu Leu 210 215 220 His Met Ser Leu Leu Tyr Ser Leu Tyr Cys Phe Glu TyrArg Trp Phe 225 230 235 240 Asn Lys Gly Ile Glu Met His Gln Arg Leu SerAsn Ile Glu Arg Asn 245 250 255 Trp Pro Tyr Tyr Phe Gly Phe Gly Leu ProLeu Ala Phe Leu Thr Ala 260 265 270 Met Gln Ser Ser Tyr Ile Ile Ser GlyCys Leu Phe Ser Ile Leu Phe 275 280 285 Pro Leu Phe Ile Ile Ser Ala AsnGlu Ala Lys Thr Pro Gly Lys Ala 290 295 300 Tyr Leu Phe Gln Leu Arg LeuPhe Ser Leu Val Val Phe Leu Ser Asn 305 310 315 320 Arg Leu Phe His LysThr Val Tyr Leu Gln Ser Ala Leu Ser Ser Ser 325 330 335 Ser Ser Ala GluLys Phe Pro Ser Pro His Pro Ser Pro Ala Lys Leu 340 345 350 Lys Ala AlaAla Gly His 355 10 315 PRT C. elegans 10 Met Val Lys Phe Gln Ile Ile AlaArg Asp Phe Tyr His Gly Phe Ile 1 5 10 15 Asn Ser Phe Lys Gly Ile ThrPhe Val Arg Arg Ile Arg Glu Glu Glu 20 25 30 Ala Lys Glu Val Lys Val GluPro Pro Lys Pro Val Glu Arg Thr Val 35 40 45 Leu Met Met Arg Arg Glu LysGln Gly Ile Phe Lys Arg Pro Pro Gly 50 55 60 Pro Pro Lys Lys Lys Asp SerPhe Leu Lys Lys Leu Tyr Gln Ile Tyr 65 70 75 80 Ala Met Asn Ile Gly PheLeu Val Leu Trp Gln Val Cys Ile Leu Ile 85 90 95 Leu Gly Leu Phe Phe SerPhe Phe Asn Arg Thr Asn Leu Gly His Asn 100 105 110 Ile Gly Tyr Ile LeuIle Ile Pro Ile Phe Phe Ala Ser Arg Ile Ile 115 120 125 Gln Ala Leu TrpPhe Ser Asp Ile Ser Gly Ala Cys Met Arg Ala Leu 130 135 140 Lys Leu ProPro Pro Pro Val Val Pro Phe Ser Ser Met Leu Ala Gly 145 150 155 160 ThrLeu Ile Ser Ala Leu His Gln Ile Phe Phe Leu Ile Gln Gly Met 165 170 175Leu Ser Gln Tyr Leu Pro Ile Pro Lys Ile Thr Pro Val Ile Val Tyr 180 185190 Leu His Met Ala Leu Leu Asn Ser Met Tyr Cys Phe Asp Tyr Phe Phe 195200 205 Asp Gly Tyr Asn Leu Ser Phe Leu Arg Arg Lys Asp Ile Phe Glu Ser210 215 220 His Trp Pro Tyr Phe Leu Gly Phe Gly Thr Pro Leu Ala Leu AlaCys 225 230 235 240 Ser Ile Ser Ser Asn Met Phe Val Asn Ser Val Ile PheAla Leu Leu 245 250 255 Phe Pro Phe Phe Ile Ile Thr Ser Tyr Pro Ala AsnTrp Asn Arg Lys 260 265 270 Tyr Glu Glu Glu Ile Pro Lys Ile Ala Phe CysArg Ile Ser Tyr Met 275 280 285 Phe Thr Glu Leu Val Gly Lys Phe Val LysSer Ile Thr Pro Thr Asn 290 295 300 Asn Pro Thr Ala Ala Arg Asn Asn AlaGln Asn 305 310 315 11 505 DNA Homo sapiens unsure (400) 11 gaatggtggagtgttctggt tcagtctcct cttgttttat cgagtattta ttcctgtgct 60 tcagtcggtaacagcccgaa ttatcggtga cccatcacta catggagatg tttggtcgtg 120 gctggaattcttcctcacgt caattttcag tgctctttgg gtgctcccct tgtttgtgct 180 tagcaaagtggtgaatgcca tttggtttca ggatatagct gacctggcat ttgaggtatc 240 agggaggaagcctcacccat tccctagtgt cagcaaaata attgctgaca tgctcttcaa 300 ccttttgctgcaggctcttt tcctcattca gggaatgttt gtgagtctct ttcccatcca 360 tcttgtcggtcagctggtta gtctcctgca tatgtccctn cttctaactt cactgtaact 420 gctttngaatantcgttggg ttcaatagga aatggcacca gcgggttgtt ctaacatagg 480 aaagggaattgggcctnact acttt 505 12 439 DNA Homo sapiens unsure (30) 12 cttcctcacgtcaattttca gtgctctttn ggtgctcccc ttntttgtgc ttagcaaagt 60 ggtgaatgccattnggtttc aggatatagc tgacctggca tttgaggtat cagggaggaa 120 gcctcacccattccctagtg tcagcaaaat aattgctgac atgctcttca accttttgct 180 gcaggctcttttcctcattc agggaatgtt tgtnagtctc tttcccatcc atcttgtngg 240 tcagctggttagtctcctgc atatgtccct tcnctactca ctgtactgct ttgaatatcg 300 ttggtttcaataaaaggaat tgaaatgcac cagcggttgt ttaacatagg aaaggaattg 360 ggccttactactttgggttt ggtttgccct tggcttttct nacagcaatg cagtcctcat 420 atattgatcagtggctgcc 439 13 415 DNA Homo sapiens unsure (347) 13 cagggaggaagcctcaccca ttccctagtg tcagcaaaat aattgctgac atgctcttca 60 accttttgctgcaggctctt ttcctcattc agggaatgtt tgtgagtctc tttcccatcc 120 atcttgtcggtcagctggtt agtctcctgc atatgtccct tctctactca ctgtactgct 180 ttgaatatcgttggttcaat aaaggaattg aaatgcacca gcggttgtct aacatagaaa 240 ggaattggccttactacttt gggtttggtt tgcccttggc ttttctcaca gcaatgcagt 300 cctcatatattatcagtggc tgccttttct ctatcctctt tcctttnttc attatcaggc 360 gccaatggaaggcaaagacc cnggggcaaa gcatattctc ttccagttgg nggcc 415 14 336 DNA Homosapiens unsure (309) 14 tccagtgttg tgcttggaat ggtggagtgt tctggttcagtctcctcttg ttttatcgag 60 tatttattcc tgtgcttcag tcggtaacag cccgaattatcggtgaccca tcactacatg 120 gagatgtttg gtcgtggctg gaattcttcc tcacgtcaattttcagtgct ctttgggtgc 180 tccccttgtt tgtgcttagc aaagtggtga atgccatttggtttcaggat atagctgacc 240 tggcatttga ggtatcaggg aggaagcctc acccattccctagttgtcag caaaataatt 300 gctgacatng ctcttncaac cttttnactt gcaggc 336 15332 DNA Homo sapiens 15 ggcatttgag gtatcaggga ggaagcctca cccattccctagtgtcagca aaataattgc 60 tgacatgctc ttcaaccttt tgctgcaggc tcttttcctcattcagggaa tgtttgtgag 120 tctctttccc atccatcttg tcggtcagct ggttagtctcctgcatatgt cccttctcta 180 ctcactgtac tgctttgaat atcgttggtt caataaaggaattgaaatgc accagcggtt 240 gtctaacata gaaaggaatt ggccttacta cttgcttgggtttgccttgg cttttctcac 300 agcaatgcag tctcatatat atcagtggtg ct 332 16 275DNA Homo sapiens 16 cagggaggaa gcctcaccca ttccctagtg tcagcaaaataattgctgac atgctcttca 60 accttttgct gcaggctctt ttcctcattc agggaatgtttgtgagtctc tttcccatcc 120 atcttgtcgg tcagctggtt agtctcctgc atatgtcccttctctactca ctgtactgct 180 ttgaatatcg ttggttcaat aaaggaattg aaatgcaccagcggttgtct aacatagaaa 240 ggaattggcc ttactacttt gggtttggtt tgccc 275 17268 DNA Homo sapiens 17 gatatagctg acctggcatt tgaggtatca gggaggaagcctcacccatt ccctagtgtc 60 agcaaaataa ttgctgacat gctcttcaac cttttgctgcaggctctttt cctcattcag 120 ggaatgtttg tgagtctctt tcccatccat cttgtcggtcagctggttag tctcctgcat 180 atgtcccttc tctactcact gtactgcttt gaatatcgttggttcaataa aggaattgaa 240 atgcaccagc ggttgtctaa catagaaa 268 18 14 DNAArtificial Sequence primer sequence 18 tttttttttt ttng 14 19 14 DNAArtificial Sequence primer sequence 19 tttttttttt ttna 14 20 14 DNAArtificial Sequence primer sequence 20 tttttttttt ttnt 14 21 14 DNAArtificial Sequence primer sequence 21 tttttttttt ttnc 14

What is claimed is:
 1. A isolated EI24 protein.
 2. The isolated EI24protein of claim 1, wherein said protein is a murine protein.
 3. Theisolated EI24 protein of claim 2, wherein said protein comprises theamino acid sequence as shown in FIG.
 2. 4. The isolated EI24 protein ofclaim 2, wherein said protein is encoded by the nucleotide sequence asshown in FIG.
 2. 5. An isolated nucleotide sequence coding for EI24protein.
 6. The isolated nucleotide sequence of claim 5, wherein saidsequence is as shown in FIG.
 2. 7. The isolated nucleotide sequence ofany one of claims 5 or 6, wherein said sequence comprises genomic DNA.8. The isolated nucleotide sequence of any one of claims 5 or 6, whereinsaid sequence comprises cDNA.
 9. The isolated nucleotide sequence of anyone of claims 5 or 6, wherein said sequence comprise RNA.
 10. Anisolated recombinant DNA molecule consisting essentially of a nucleotidesequence that codes for EI24 protein.
 11. The isolated recombinant DNAmolecule of claim 10, wherein said nucleotide sequence is as shown inFIG.
 2. 12. The isolated recombinant DNA molecule of claim 11, whereinsaid molecule is a vector.
 13. A vector consisting essentially of arecombinant DNA molecule encoding EI24 protein, wherein said vectorexpresses a sequence that codes for EI24 in said recombinant DNAmolecule.
 14. A vector comprising a recombinant DNA molecule encodingEI24 protein, wherein said vector expresses an antisense RNA of saidrecombinant molecule.
 15. The vector of claim 13, wherein saidrecombinant DNA molecule consists essentially of a nucleotide sequenceas shown in FIG.
 2. 16. A host cell transformed with the vector of anyone of claims 13 or
 15. 17. The host cell of claim 11, wherein said hostcell is a mammalian cell.
 18. A method for producing isolated EI24polypeptide, comprising: (a) constructing the vector of claim 13; (b)transforming a suitable host cell with said vector of Step (a); (c)culturing said host cell under conditions which allow the expression ofsaid EI24 polypeptide by said host cell; and (d) isolating said EI24polypeptide expressed by said host cell of Step (c); wherein isolatedsubstantially pure EI24 polypeptide is produced.
 19. The methodaccording to claim 18, wherein said host cell is a mammalian cell. 20.An antibody raised against the EI24 protein of claim
 4. 21. The antibodyof claim 20, wherein said antibody is selected from the group consistingof: a polyclonal antibody, a monoclonal antibody, an anti-idiotypicantibody, and an anti-anti-idiotypic antibody.
 22. The antibody of claim21, wherein said antibody is detectably labeled.
 23. The antibody ofclaim 22, wherein said antibody is detectably labeled with a detectablelabel selected from the group consisting of: a radio label, an enzymelabel, a co-factor label, a fluorescent label, a paramagnetic label, achemiluminescent label, and a metal label.
 24. A detectably labelednucleotide probe, comprising a first nucleotide sequence which issubstantially complementary to a second nucleotide sequence thatspecifically codes for the EI24 protein of claim
 4. 25. A pharmaceuticalcomposition comprising the EI24 protein of claim 4 and apharmaceutically acceptable carrier.
 26. A method for inducing apoptosisin a cell comprising introducing into a nonapoptotic cell an amount ofthe EI24 protein of claim 4 effective to induce apoptosis in said cell.27. The method of claim 26, wherein said EI24 protein is introduced byexpressing a nucleotide encoding EI24 in said nonapoptotic cell.
 28. Amethod for detecting the expression of the EI24 gene in a biologicalsample comprising the steps of identifying the presence of mRNA encodingEI24.
 29. The method according to claim 28 wherein the method foridentifying said mRNA is Northern blotting.
 30. A method of identifyingEI24 mRNA comprising the steps of: (a) obtaining a cell sample; (b)obtaining mRNA from the cell sample; (c) performing a polymerase chainreaction on said mRNA using the primers corresponding to the uniqueregions of EI24; and  detecting the presence of the products of thepolymerase chain reaction.
 31. A method of assaying for interactionsbetween EI24 and proteins which bind specifically to EI24 comprising thesteps of: (a) contracting purified EI24 with cell lysates containing aprotein which binds to EI24, under conditions which allow binding ofsaid protein to said EI24; (b) isolating said EI24; (c) contacting theisolated EI24 of step (b) with a binding partner specific for theprotein, under conditions which allow binding of said binding partner tosaid EI24; (d) measuring the amount of said binding partner bound tosaid EI24 protein.
 32. The method of claim 32 wherein said cell lysateis a yeast lysate.
 33. The method of claim 32 wherein said bindingpartner is an antibody.
 34. A cDNA clone selected from the groupconsisting of pKSEI24 cl.11 and pKSEI24 1-2.